Genetically programmed expression of selectively sulfated proteins in eubacteria

ABSTRACT

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid sulfotyrosine into proteins produced in eubacterial host cells such as  E. coli . The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid sulfotyrosine and translation systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of: U.S. ProvisionalAppl. Ser. No. 60/846,519, filed Sep. 21, 2006; and U.S. ProvisionalAppl. Ser. No. 60/855,210, filed Oct. 28, 2006, the disclosures of whichare both hereby incorporated by reference in their entirety for allpurposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support from the NationalInstitutes of Health under Grant No. GM62159. The government may havecertain rights to this invention.

FIELD OF THE INVENTION

The invention is in the field of translation biochemistry. The inventionrelates to compositions and methods for making and using orthogonaltRNAs, orthogonal aminoacyl-tRNA synthetases, and pairs thereof, thatincorporate unnatural amino acids into proteins. The invention alsorelates to methods of producing proteins in cells using such pairs andproteins made by the methods.

BACKGROUND OF THE INVENTION

Tyrosine sulfation is a common post-translational modification insecreted and membrane-bound proteins (Kehoe and Bertozzi, “Tyrosinesulfation: a modulator of extracellular protein-protein interactions,”Chem Biol 7:R57-61 (2000)). Although we are only beginning to understandthe extent of its biological function, sulfotyrosine has already beenidentified in several protein-protein interaction paradigms. Forexample, tyrosine sulfation plays a determining role in chemokinebinding to the chemokine receptors CCR2 (Preobrazhensky et al.,“Monocyte chemotactic protein-1 receptor CCR2B is a glycoprotein thathas tyrosine sulfation in a conserved extracellular N-terminal region” JImmunol 165:5295-5303 (2000)), CCR5 (Farzan et al., “Tyrosine sulfationof the amino terminus of CCR5 facilitates HIV-1 entry” Cell 96:667-676(1999)), CXCR4 (Farzan et al., “The role of post-translationalmodifications of the CXCR4 amino terminus in stromal-derived factor 1alpha association and HIV-1 entry,” J Biol Chem 277:29484-29489 (2002);Veldkamp et al., “Recognition of a CXCR4 sulfotyrosine by the chemokinestromal cell-derived factor-1alpha (SDF-1alpha/CXCL12),” J Mol Biol359:1400-1409 (2006)) and CX₃CR1 (Fong et al., “CX3CR1 tyrosinesulfation enhances fractalkine-induced cell adhesion,” J Biol Chem277:19418-19423 (2002)). Similarly, leukocyte rolling under hydrodynamicshear stresses requires sulfation of PSGL-1 for proper binding andadhesion (Somers et al., “Insights into the molecular basis of leukocytetethering and rolling revealed by structures of P- and E-selectin boundto SLe(X) and PSGL-1,” Cell 103:467-479 (2000)). Tyrosine sulfation isalso involved in the coagulation cascade, having been identified inseveral clotting factors as well as in natural thrombin inhibitors suchas the leech-secreted anticoagulant hirudin (Dong et al., “Tyrosinesulfation of the glycoprotein Ib-IX complex: identification of sulfatedresidues and effect on ligand binding,” Biochemistry 33:13946-13953(1994); Bagdy et al., “Hirudin,” Methods Enzymol 45:669-678 (1976)). Inaddition, it was recently discovered that tyrosine sulfation on anantibody variable loop region is responsible for the neutralizingactivity of a subset of CD4-induced HIV-1 antibodies, thus demonstratingthe ability of sulfotyrosine to augment antibody-antigen affinity (Choeet al., “Tyrosine sulfation of human antibodies contributes torecognition of the CCR5 binding region of HIV-1 gp120,” Cell 114:161-170(2003); Xiang et al., “Functional mimicry of a human immunodeficiencyvirus type 1 coreceptor by a neutralizing monoclonal antibody,” J Virol79:6068-6077 (2005)).

A major obstacle to determining the functions of sulfation in the over60 known and over 2100 predicted proteins containing sulfotyrosine(based on a study of mouse protein sequences) is the ability tosynthesize selectively sulfated proteins (Moore, “The biology andenzymology of protein tyrosine O-sulfation,” J Biol Chem 278:24243-24246(2003)). Current methods rely on standard peptide synthesis or in vitroenzymatic sulfation (Veldkamp et al., “Recognition of a CXCR4sulfotyrosine by the chemokine stromal cell-derived factor-1 alpha(SDF-1alpha/CXCL12),” J Mol Biol 359:1400-1409 (2006); Kirano et al.,“Total synthesis of porcine cholecystokinin-33 (CCK-33),” J. Chem. Soc.,Chem. Commun., 323-325 (1987); Muramatsu et al., “Enzymic O-sulfation oftyrosine residues in hirudins by sulfotransferase from EubacteriumA-44,” Eur J Biochem 223:243-248 (1994); Young and Kiessling, “Astrategy for the synthesis of sulfated peptides,” Angew Chem Int Ed Engl41:3449-3451 (2002)); however, both lack generality: the former islimited by length restrictions and the tendency towards sulfotyrosinedesulfation under acidic conditions; the latter is limited by theavailability of accessory sulfotransferases and their associatedrecognition sequence constraints.

The direct incorporation of a genetically encoded sulfotyrosineunnatural amino acid at defined sites in proteins directly in livingorganisms would overcome the limitations described above. The directincorporation of sulfotyrosine will greatly facilitate the study ofsulfation events in the regulation of biological processes and will alsoallow for the creation of sulfated antibody and peptide libraries ofsignificant diversity. Furthermore, the ability to produce a sulfatedform of the protein hirudin has immediate clinical application for useas an improved anticoagulant (improved relative to the non-sulfatedform). What are needed in the art are new strategies for incorporationof sulfotyrosine unnatural amino acid into proteins.

A general methodology has been developed for the in vivo site-specificincorporation of diverse unnatural amino acids into proteins in bothprokaryotic and eukaryotic organisms. These methods rely on orthogonalprotein translation components that recognize a suitable selector codonto insert a desired unnatural amino acid at a defined position duringpolypeptide translation in vivo. These methods utilize an orthogonaltRNA (O-tRNA) that recognizes a selector codon, and where acorresponding specific orthogonal aminoacyl-tRNA synthetase (an O-RS)charges the O-tRNA with the unnatural amino acid. These components donot cross-react with any of the endogenous tRNAs, RSs, amino acids orcodons in the host organism (i.e., it must be orthogonal). The use ofsuch orthogonal tRNA-RS pairs has made it possible to genetically encodea large number of structurally diverse unnatural amino acids.

The practice of using orthogonal translation systems that are suitablefor making proteins that comprise one or more unnatural amino acid isgenerally known in the art, as are the general methods for producingorthogonal translation systems. For example, see InternationalPublication Numbers WO 2002/086075, entitled “METHODS AND COMPOSITIONFOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;”WO 2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINOACIDS;” WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC GENETICCODE;” WO 2005/019415, filed Jul. 7, 2004; WO 2005/007870, filed Jul. 7,2004; WO 2005/007624, filed Jul. 7, 2004 and WO 2006/110182, filed Oct.27, 2005, entitled “ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVOINCORPORATION OF UNNATURAL AMINO ACIDS.” Each of these applications isincorporated herein by reference in its entirety. For additionaldiscussion of orthogonal translation systems that incorporate unnaturalamino acids, and methods for their production and use, see also, Wangand Schultz, “Expanding the Genetic Code,” Chem. Commun. (Camb.) 1:1-11(2002); Wang and Schultz “Expanding the Genetic Code,” Angewandte ChemieInt. Ed., 44(1):34-66 (2005); Xie and Schultz, “An Expanding GeneticCode,” Methods 36(3):227-238 (2005); Xie and Schultz, “Adding AminoAcids to the Genetic Repertoire,” Curr. Opinion in Chemical Biology9(6):548-554 (2005); Wang et al., “Expanding the Genetic Code,” Annu.Rev. Biophys. Biomol. Struct., 35:225-249 (2006; epub Jan. 13, 2006);and Xie and Schultz, “A chemical toolkit for proteins—an expandedgenetic code,” Nat. Rev. Mol. Cell Biol., 7(10):775-782 (2006; epub Aug.23, 2006).

There is a need in the art for the development of orthogonal translationcomponents that incorporate sulfotyrosine unnatural amino acid intoproteins, where the unnatural amino acid can be incorporated at anydefined position. The invention described herein fulfills these andother needs, as will be apparent upon review of the followingdisclosure.

SUMMARY OF THE INVENTION

Although tyrosine sulfation is a post-translational modificationwidespread across multicellular eukaryotes (Moore, “The biology andenzymology of protein tyrosine O-sulfation,” J Biol Chem 278:24243-24246(2003)), its biological functions remain largely unknown. This is inpart due to the difficulties associated with the synthesis ofselectively sulfated proteins. The invention provides for the selectiveincorporation of sulfotyrosine into proteins in bacteria by geneticallyencoding the modified amino acid in response to the amber nonsensecodon, TAG. Moreover, it is demonstrated that sulfo-hirudin, previouslyinaccessible through recombinant methods, can be directly expressed inE. coli using this strategy. As described herein, kinetic analyses showa greater than 10-fold enhancement in affinity towards human thrombin bysulfo-hirudin over desulfo-hirudin, an observation that offers clinicaladvantages for sulfo-hirudin in its use as an anticoagulant (Di Nisio etal., “Direct thrombin inhibitors,” N Engl J Med 353:1028-1040 (2005)).This general approach to the biosynthesis of sulfated proteinsfacilitates further study and application of the emergingpost-translational modification, tyrosine sulfation.

The invention provides compositions and methods for incorporating theunnatural amino acid sulfotyrosine into a growing polypeptide chain inresponse to a selector codon, e.g., an amber stop codon, in vivo (e.g.,in a host cell). These compositions include pairs of orthogonal-tRNAs(O-tRNAs) and orthogonal aminoacyl-tRNA synthetases (O-RSs) that do notinteract with the host cell translation machinery. That is to say, theO-tRNA is not charged (or not charged to a significant level) with anamino acid (natural or unnatural) by an endogenous host cellaminoacyl-tRNA synthetase. Similarly, the O-RSs provided by theinvention do not charge any endogenous tRNA with an amino acid (naturalor unnatural) to a significant or detectable level. These novelcompositions permit the production of large quantities of proteinshaving translationally incorporated sulfotyrosine.

In some aspects, the invention provides translation systems. Thesesystems comprise a first orthogonal aminoacyl-tRNA synthetase (O-RS), afirst orthogonal tRNA (O-tRNA), and a first unnatural amino acid that issulfotyrosine, where the first O-RS preferentially aminoacylates thefirst O-tRNA with the first unnatural amino acid sulfotyrosine. In someaspects, the O-RS preferentially aminoacylates the O-tRNA with saidsulfotyrosine with an efficiency that is at least 50% of the efficiencyobserved for a translation system comprising that same O-tRNA, thesulfotyrosine, and an aminoacyl-tRNA synthetase comprising the aminoacid sequence of SEQ ID NO: 4, 6, 8 or 10.

The translation systems can use components derived from a variety ofsources. In one embodiment, the first O-RS is derived from aMethanococcus jannaschii aminoacyl-tRNA synthetase, e.g., a wild-typeMethanococcus jannaschii tyrosyl-tRNA synthetase. The O-RS used in thesystem can comprise the amino acid sequence of SEQ ID NOS: 4, 6, 8 or10, and conservative variants of that sequence. In some embodiments, theO-tRNA is an amber suppressor tRNA. In some embodiments, the O-tRNAcomprises or is encoded by SEQ ID NO: 1.

In some aspects, the translation system further comprises a nucleic acidencoding a protein of interest, where the nucleic acid has at least oneselector codon that is recognized by the O-tRNA.

In some aspects, the translation system incorporates a second orthogonalpair (that is, a second O-RS and a second O-tRNA) that utilizes a secondunnatural amino acid, so that the system is now able to incorporate atleast two different unnatural amino acids at different selected sites ina polypeptide. In this dual system, the second O-RS preferentiallyaminoacylates the second O-tRNA with a second unnatural amino acid thatis different from the first unnatural amino acid, and the second O-tRNArecognizes a selector codon that is different from the selector codonrecognized by the first O-tRNA.

In some embodiments, the translation system resides in a host cell (andincludes the host cell). The host cell used is not particularly limited,as long as the O-RS and O-tRNA retain their orthogonality in their hostcell environment. The host cell can be a eubacterial cell, such as E.coli. The host cell can comprise one or more polynucleotides that encodecomponents of the translation system, including the O-RS or O-tRNA. Insome embodiments, the polynucleotide encoding the O-RS comprises anucleotide sequence of SEQ ID NO: 5, 7, 9 or 11.

The invention also provides methods for producing proteins having one ormore unnatural amino acids at selected positions. These methods utilizethe translation systems described above. Generally, these methods startwith the step of providing a translation system comprising: (i) a firstunnatural amino acid that is the unnatural amino acid sulfotyrosine;(ii) a first orthogonal aminoacyl-tRNA synthetase (O-RS); (iii) a firstorthogonal tRNA (O-tRNA), wherein the O-RS preferentially aminoacylatesthe O-tRNA with the unnatural amino acid; and, (iv) a nucleic acidencoding the protein, where the nucleic acid comprises at least oneselector codon that is recognized by the first O-tRNA. The method thenincorporates the unnatural amino acid at the selected position in theprotein during translation of the protein in response to the selectorcodon, thereby producing the protein comprising the unnatural amino acidat the selected position. In some aspects of these methods, the O-RSpreferentially aminoacylates the O-tRNA with the sulfotyrosine with anefficiency that is at least 50% of the efficiency observed for atranslation system comprising that same O-tRNA, the sulfotyrosine, andan aminoacyl-tRNA synthetase comprising the amino acid sequence of SEQID NO: 4, 6, 8 or 10. In some aspects, the methods are used to producethe sulfated form of hirudin.

These methods can be widely applied using a variety of reagents andsteps. In some embodiments, a polynucleotide encoding the O-RS isprovided. In some embodiments, an O-RS derived from a Methanococcusjannaschii aminoacyl-tRNA synthetase is provided, for example, awild-type Methanococcus jannaschii tyrosyl-tRNA synthetase can beprovided. In some embodiments, the providing step includes providing anO-RS comprising an amino acid sequence of SEQ ID NO: 4, 6, 8 or 10, andconservative variants thereof.

In some embodiments of these methods, the providing a translation systemstep comprises mutating an amino acid binding pocket of a wild-typeaminoacyl-tRNA synthetase by site-directed mutagenesis, and selecting aresulting O-RS that preferentially aminoacylates the O-tRNA with theunnatural amino acid. The selecting step can comprises positivelyselecting and negatively selecting for the O-RS from a pool of resultingaminoacyl-tRNA synthetase molecules following site-directed mutagenesis.In some embodiments, the providing step furnishes a polynucleotideencoding the O-tRNA, e.g., an O-tRNA that is an amber suppressor tRNA,or an O-tRNA that comprises or is encoded by a polynucleotide of SEQ IDNO: 1. In these methods, the providing step can also furnish a nucleicacid comprising an amber selector codon that is utilized by thetranslation system.

These methods can also be modified to incorporate more than oneunnatural amino acid into a protein. In those methods, a secondorthogonal translation system is employed in conjunction with the firsttranslation system, where the second system has different amino acid andselector codon specificities. For example, the providing step caninclude providing a second O-RS and a second O-tRNA, where the secondO-RS preferentially aminoacylates the second O-tRNA with a secondunnatural amino acid that is different from the first unnatural aminoacid, and where the second O-tRNA recognizes a selector codon in thenucleic acid that is different from the selector codon recognized by thefirst O-tRNA.

The methods for producing a protein with an unnatural amino acid canalso be conducted in the context of a host cell. In these cases, a hostcell is provided, where the host cell comprises the unnatural aminoacid, the O-RS, the O-tRNA and the nucleic acid with at least oneselector codon that encodes the protein, and where culturing the hostcell results in incorporating the unnatural amino acid. In someembodiments, the providing step comprises providing a eubacterial hostcell (e.g., E. coli). In some embodiments, the providing step includesproviding a host cell that contains a polynucleotide encoding the O-RS.For example, the polynucleotide encoding the O-RS can comprise anucleotide sequence of SEQ ID NO: 5, 7, 9 or 11. In some embodiments,the step of providing a translation system is accomplished by providinga cell extract.

The invention also provides a variety of compositions, including nucleicacids and proteins. The nature of the composition is not particularlylimited, other than the composition comprises the specified nucleic acidor protein. The compositions of the invention can comprise any number ofadditional components of any nature.

For example, the invention provides compositions comprising O-RSpolypeptides, where the polypeptides comprise the amino acid sequence ofSEQ ID NO: 4, 6, 8 or 10, or a conservative variant thereof. In someaspects, the conservative variant polypeptide aminoacylates a cognateorthogonal tRNA (O-tRNA) with an unnatural amino acid with an efficiencythat is at least 50% of the efficiency observed for a translation systemcomprising the O-tRNA, the unnatural amino acid, and an aminoacyl-tRNAsynthetase comprising the amino acid sequence of SEQ ID NO: 4, 6, 8 or10. The invention also provides polynucleotides that encode any of thesepolypeptides above. In some embodiments, these polynucleotides cancomprise a nucleotide sequence of SEQ ID NO: 5, 7, 9 or 11. In someembodiments, the polypeptides are in a cell.

The invention also provides polynucleotide compositions comprising anucleotide sequence of SEQ ID NO: 5, 7, 9 or 11. In some embodiments,the invention provides vectors comprising the polynucleotides, e.g.,expression vectors. In some embodiments, the invention provides cellscomprising a vector described above.

DEFINITIONS

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. As used in this specificationand the appended claims, the singular forms “a”, “an” and “the” includeplural referents unless the content clearly dictates otherwise. Thus,for example, reference to “a cell” includes combinations of two or morecells; reference to “a polynucleotide” includes, as a practical matter,many copies of that polynucleotide.

Unless defined herein and below in the reminder of the specification,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which theinvention pertains.

Orthogonal: As used herein, the term “orthogonal” refers to a molecule(e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl-tRNAsynthetase (O-RS)) that functions with endogenous components of a cellwith reduced efficiency as compared to a corresponding molecule that isendogenous to the cell or translation system, or that fails to functionwith endogenous components of the cell. In the context of tRNAs andaminoacyl-tRNA synthetases, orthogonal refers to an inability or reducedefficiency, e.g., less than 20% efficiency, less than 10% efficiency,less than 5% efficiency, or less than 1% efficiency, of an orthogonaltRNA to function with an endogenous tRNA synthetase compared to anendogenous tRNA to function with the endogenous tRNA synthetase, or ofan orthogonal aminoacyl-tRNA synthetase to function with an endogenoustRNA compared to an endogenous tRNA synthetase to function with theendogenous tRNA. The orthogonal molecule lacks a functionally normalendogenous complementary molecule in the cell. For example, anorthogonal tRNA in a cell is aminoacylated by any endogenous RS of thecell with reduced or even zero efficiency, when compared toaminoacylation of an endogenous tRNA by the endogenous RS. In anotherexample, an orthogonal RS aminoacylates any endogenous tRNA a cell ofinterest with reduced or even zero efficiency, as compared toaminoacylation of the endogenous tRNA by an endogenous RS. A secondorthogonal molecule can be introduced into the cell that functions withthe first orthogonal molecule. For example, an orthogonal tRNA/RS pairincludes introduced complementary components that function together inthe cell with an efficiency (e.g., 45% efficiency, 50% efficiency, 60%efficiency, 70% efficiency, 75% efficiency, 80% efficiency, 90%efficiency, 95% efficiency, or 99% or more efficiency) as compared tothat of a control, e.g., a corresponding tRNA/RS endogenous pair, or anactive orthogonal pair (e.g., a tyrosyl orthogonal tRNA/RS pair).

Orthogonal tyrosyl-tRNA: As used herein, an orthogonal tyrosyl-tRNA(tyrosyl-O-tRNA) is a tRNA that is orthogonal to a translation system ofinterest, where the tRNA is: (1) identical or substantially similar to anaturally occurring tyrosyl-tRNA, (2) derived from a naturally occurringtyrosyl-tRNA by natural or artificial mutagenesis, (3) derived by anyprocess that takes a sequence of a wild-type or mutant tyrosyl-tRNAsequence of (1) or (2) into account, (4) homologous to a wild-type ormutant tyrosyl-tRNA; (5) homologous to any example tRNA that isdesignated as a substrate for a tyrosyl-tRNA synthetase in FIG. 7, or(6) a conservative variant of any example tRNA that is designated as asubstrate for a tyrosyl-tRNA synthetase in FIG. 7. The tyrosyl-tRNA canexist charged with an amino acid, or in an uncharged state. It is alsoto be understood that a “tyrosyl-O-tRNA” optionally is charged(aminoacylated) by a cognate synthetase with an amino acid other thantyrosine, respectively, e.g., with an unnatural amino acid. Indeed, itwill be appreciated that a tyrosyl-O-tRNA of the invention isadvantageously used to insert essentially any amino acid, whethernatural or unnatural, into a growing polypeptide, during translation, inresponse to a selector codon.

Orthogonal tyrosyl amino acid synthetase: As used herein, an orthogonaltyrosyl amino acid synthetase (tyrosyl-O-RS) is an enzyme thatpreferentially aminoacylates the tyrosyl-O-tRNA with an amino acid in atranslation system of interest. The amino acid that the tyrosyl-O-RSloads onto the tyrosyl-O-tRNA can be any amino acid, whether natural,unnatural or artificial, and is not limited herein. The synthetase isoptionally the same as or homologous to a naturally occurring tyrosylamino acid synthetase, or the same as or homologous to a synthetasedesignated as an O-RS in FIG. 7. For example, the O-RS can be aconservative variant of a tyrosyl-O-RS of FIG. 7, and/or can be at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in sequence toan O-RS of FIG. 7.

Cognate: The term “cognate” refers to components that function together,or have some aspect of specificity for each other, e.g., an orthogonaltRNA and an orthogonal aminoacyl-tRNA synthetase. The components canalso be referred to as being complementary.

Preferentially aminoacylates: As used herein in reference to orthogonaltranslation systems, an O-RS “preferentially aminoacylates” a cognateO-tRNA when the O-RS charges the O-tRNA with an amino acid moreefficiently than it charges any endogenous tRNA in an expression system.That is, when the O-tRNA and any given endogenous tRNA are present in atranslation system in approximately equal molar ratios, the O-RS willcharge the O-tRNA more frequently than it will charge the endogenoustRNA. Preferably, the relative ratio of O-tRNA charged by the O-RS toendogenous tRNA charged by the O-RS is high, preferably resulting in theO-RS charging the O-tRNA exclusively, or nearly exclusively, when theO-tRNA and endogenous tRNA are present in equal molar concentrations inthe translation system. The relative ratio between O-tRNA and endogenoustRNA that is charged by the O-RS, when the O-tRNA and O-RS are presentat equal molar concentrations, is greater than 1:1, preferably at leastabout 2:1, more preferably 5:1, still more preferably 10:1, yet morepreferably 20:1, still more preferably 50:1, yet more preferably 75:1,still more preferably 95:1, 98:1, 99:1, 100:1, 500:1, 1,000:1, 5,000:1or higher.

The O-RS “preferentially aminoacylates an O-tRNA with an unnatural aminoacid” when (a) the O-RS preferentially aminoacylates the O-tRNA comparedto an endogenous tRNA, and (b) where that aminoacylation is specific forthe unnatural amino acid, as compared to aminoacylation of the O-tRNA bythe O-RS with any natural amino acid. That is, when the unnatural andnatural amino acids are present in equal molar amounts in a translationsystem comprising the O-RS and O-tRNA, the O-RS will load the O-tRNAwith the unnatural amino acid more frequently than with the naturalamino acid. Preferably, the relative ratio of O-tRNA charged with theunnatural amino acid to O-tRNA charged with the natural amino acid ishigh. More preferably, O-RS charges the O-tRNA exclusively, or nearlyexclusively, with the unnatural amino acid. The relative ratio betweencharging of the O-tRNA with the unnatural amino acid and charging of theO-tRNA with the natural amino acid, when both the natural and unnaturalamino acids are present in the translation system in equal molarconcentrations, is greater than 1:1, preferably at least about 2:1, morepreferably 5:1, still more preferably 10:1, yet more preferably 20:1,still more preferably 50:1, yet more preferably 75:1, still morepreferably 95:1, 98:1, 99:1, 100:1, 500:1, 1,000:1, 5,000:1 or higher.

Selector codon: The term “selector codon” refers to codons recognized bythe O-tRNA in the translation process and not recognized by anendogenous tRNA. The O-tRNA anticodon loop recognizes the selector codonon the mRNA and incorporates its amino acid, e.g., an unnatural aminoacid, at this site in the polypeptide. Selector codons can include,e.g., nonsense codons, such as, stop codons, e.g., amber, ochre, andopal codons; four or more base codons; rare codons; codons derived fromnatural or unnatural base pairs and/or the like.

Suppressor tRNA: A suppressor tRNA is a tRNA that alters the reading ofa messenger RNA (mRNA) in a given translation system, typically byallowing the incorporation of an amino acid in response to a stop codon(i.e., “read-through”) during the translation of a polypeptide. In someaspects, a selector codon of the invention is a suppressor codon, e.g.,a stop codon (e.g., an amber, ocher or opal codon), a four base codon, arare codon, etc.

Suppression activity: As used herein, the term “suppression activity”refers, in general, to the ability of a tRNA (e.g., a suppressor tRNA)to allow translational read-through of a codon (e.g., a selector codonthat is an amber codon or a 4-or-more base codon) that would otherwiseresult in the termination of translation or mistranslation (e.g.,frame-shifting). Suppression activity of a suppressor tRNA can beexpressed as a percentage of translational read-through activityobserved compared to a second suppressor tRNA, or as compared to acontrol system, e.g., a control system lacking an O-RS.

The present invention provides various methods by which suppressionactivity can be quantitated. Percent suppression of a particular O-tRNAand O-RS against a selector codon (e.g., an amber codon) of interestrefers to the percentage of activity of a given expressed test marker(e.g., LacZ), that includes a selector codon, in a nucleic acid encodingthe expressed test marker, in a translation system of interest, wherethe translation system of interest includes an O-RS and an O-tRNA, ascompared to a positive control construct, where the positive controllacks the O-tRNA, the O-RS and the selector codon. Thus, for example, ifan active positive control marker construct that lacks a selector codonhas an observed activity of X in a given translation system, in unitsrelevant to the marker assay at issue, then percent suppression of atest construct comprising the selector codon is the percentage of X thatthe test marker construct displays under essentially the sameenvironmental conditions as the positive control marker was expressedunder, except that the test marker construct is expressed in atranslation system that also includes the O-tRNA and the O-RS.Typically, the translation system expressing the test marker alsoincludes an amino acid that is recognized by the O-RS and O-tRNA.Optionally, the percent suppression measurement can be refined bycomparison of the test marker to a “background” or “negative” controlmarker construct, which includes the same selector codon as the testmarker, but in a system that does not include the O-tRNA, O-RS and/orrelevant amino acid recognized by the O-tRNA and/or O-RS. This negativecontrol is useful in normalizing percent suppression measurements toaccount for background signal effects from the marker in the translationsystem of interest.

Suppression efficiency can be determined by any of a number of assaysknown in the art. For example, a β-galactosidase reporter assay can beused, e.g., a derivatived lacZ plasmid (where the construct has aselector codon n the lacZ nucleic acid sequence) is introduced intocells from an appropriate organism (e.g., an organism where theorthogonal components can be used) along with plasmid comprising anO-tRNA of the invention. A cognate synthetase can also be introduced(either as a polypeptide or a polynucleotide that encodes the cognatesynthetase when expressed). The cells are grown in media to a desireddensity, e.g., to an OD₆₀₀ of about 0.5, and β-galactosidase assays areperformed, e.g., using the BetaFluor™ β-Galactosidase Assay Kit(Novagen). Percent suppression can be calculated as the percentage ofactivity for a sample relative to a comparable control, e.g., the valueobserved from the derivatized lacZ construct, where the construct has acorresponding sense codon at desired position rather than a selectorcodon.

Translation system: The term “translation system” refers to thecomponents that incorporate an amino acid into a growing polypeptidechain (protein). Components of a translation system can include, e.g.,ribosomes, tRNAs, synthetases, mRNA and the like. The O-tRNA and/or theO-RSs of the invention can be added to or be part of an in vitro or invivo translation system, e.g., in a non-eukaryotic cell, e.g., abacterium (such as E. coli), or in a eukaryotic cell, e.g., a yeastcell, a mammalian cell, a plant cell, an algae cell, a fungus cell, aninsect cell, and/or the like.

Unnatural amino acid: As used herein, the term “unnatural amino acid”refers to any amino acid, modified amino acid, and/or amino acidanalogue, that is not one of the 20 common naturally occurring aminoacids or seleno cysteine or pyrrolysine. For example, the unnaturalamino acid sulfotyrosine; see FIG. 1) finds use with the invention.

Derived from: As used herein, the term “derived from” refers to acomponent that is isolated from or made using a specified molecule ororganism, or information from the specified molecule or organism. Forexample, a polypeptide that is derived from a second polypeptide caninclude an amino acid sequence that is identical or substantiallysimilar to the amino acid sequence of the second polypeptide. In thecase of polypeptides, the derived species can be obtained by, forexample, naturally occurring mutagenesis, artificial directedmutagenesis or artificial random mutagenesis. The mutagenesis used toderive polypeptides can be intentionally directed or intentionallyrandom, or a mixture of each. The mutagenesis of a polypeptide to createa different polypeptide derived from the first can be a random event(e.g., caused by polymerase infidelity) and the identification of thederived polypeptide can be made by appropriate screening methods, e.g.,as discussed herein. Mutagenesis of a polypeptide typically entailsmanipulation of the polynucleotide that encodes the polypeptide.

Positive selection or screening marker: As used herein, the term“positive selection or screening marker” refers to a marker that, whenpresent, e.g., expressed, activated or the like, results inidentification of a cell, which comprises the trait, e.g., a cell withthe positive selection marker, from those without the trait.

Negative selection or screening marker: As used herein, the term“negative selection or screening marker” refers to a marker that, whenpresent, e.g., expressed, activated, or the like, allows identificationof a cell that does not comprise a selected property or trait (e.g., ascompared to a cell that does possess the property or trait).

Reporter: As used herein, the term “reporter” refers to a component thatcan be used to identify and/or select target components of a system ofinterest. For example, a reporter can include a protein, e.g., anenzyme, that confers antibiotic resistance or sensitivity (e.g.,β-lactamase, chloramphenicol acetyltransferase (CAT), and the like), afluorescent screening marker (e.g., green fluorescent protein (e.g.,(GFP), YFP, EGFP, RFP, etc.), a luminescent marker (e.g., a fireflyluciferase protein), an affinity based screening marker, or positive ornegative selectable marker genes such as lacZ, β-gal/lacZ(β-galactosidase), ADH (alcohol dehydrogenase), his3, ura3, leu2, lys2,or the like.

Eukaryote: As used herein, the term “eukaryote” refers to organismsbelonging to the Kingdom Eucarya. Eukaryotes are generallydistinguishable from prokaryotes by their typically multicellularorganization (but not exclusively multicellular, for example, yeast),the presence of a membrane-bound nucleus and other membrane-boundorganelles, linear genetic material (i.e., linear chromosomes), theabsence of operons, the presence of introns, message capping and poly-AmRNA, and other biochemical characteristics, such as a distinguishingribosomal structure. Eukaryotic organisms include, for example, animals(e.g., mammals, insects, reptiles, birds, etc.), ciliates, plants (e.g.,monocots, dicots, algae, etc.), fungi, yeasts, flagellates,microsporidia, protists, etc.

Prokaryote: As used herein, the term “prokaryote” refers to organismsbelonging to the Kingdom Monera (also termed Procarya). Prokaryoticorganisms are generally distinguishable from eukaryotes by theirunicellular organization, asexual reproduction by budding or fission,the lack of a membrane-bound nucleus or other membrane-bound organelles,a circular chromosome, the presence of operons, the absence of introns,message capping and poly-A mRNA, and other biochemical characteristics,such as a distinguishing ribosomal structure. The Prokarya includesubkingdoms Eubacteria and Archaea (sometimes termed “Archaebacteria”).Cyanobacteria (the blue green algae) and mycoplasma are sometimes givenseparate classifications under the Kingdom Monera.

Bacteria: As used herein, the terms “bacteria” and “eubacteria” refer toprokaryotic organisms that are distinguishable from Archaea. Similarly,Archaea refers to prokaryotes that are distinguishable from eubacteria.Eubacteria and Archaea can be distinguished by a number morphologicaland biochemical criteria. For example, differences in ribosomal RNAsequences, RNA polymerase structure, the presence or absence of introns,antibiotic sensitivity, the presence or absence of cell wallpeptidoglycans and other cell wall components, the branched versusunbranched structures of membrane lipids, and the presence/absence ofhistones and histone-like proteins are used to assign an organism toEubacteria or Archaea.

Examples of Eubacteria include Escherichia coli, Therinus thermophilus,Bacillus subtilis and Bacillus stearothermophilus. Example of Archaeainclude Methanococcus jannaschii (Mj), Methanosarcina mazei (Mm),Methanobacterium thermoautotrophicum (Mt), Methanococcus maripaludis,Methanopyrus kandleri, Halobacterium such as Haloferax volcanii andHalobacterium species NRC-1, Archaeoglobus fulgidus (Af), Pyrococcusfuriosus (Pf), Pyrococcus horikoshii (Ph), Pyrobaculum aerophilum,Pyrococcus abyssi, Sulfolobus solfataricus (Ss), Sulfolobus tokodaii,Aeuropyrum pernix (Ap), Thermoplasma acidophilum and Thermoplasmavolcanium.

Conservative variant: As used herein, the term “conservative variant,”in the context of a translation component, refers to a translationcomponent, e.g., a conservative variant O-tRNA or a conservative variantO-RS, that functionally performs similar to a base component that theconservative variant is similar to, e.g., an O-tRNA or O-RS, havingvariations in the sequence as compared to a reference O-tRNA or O-RS.For example, an O-RS, or a conservative variant of that O-RS, willaminoacylate a cognate O-tRNA with an unnatural amino acid, e.g.,sulfotyrosine. In this example, the O-RS and the conservative variantO-RS do not have the same amino acid sequences. The conservative variantcan have, e.g., one variation, two variations, three variations, fourvariations, or five or more variations in sequence, as long as theconservative variant is still complementary to (e.g., functions with)the cognate corresponding O-tRNA or O-RS.

In some embodiments, a conservative variant O-RS comprises one or moreconservative amino acid substitutions compared to the O-RS from which itwas derived. In some embodiments, a conservative variant O-RS comprisesone or more conservative amino acid substitutions compared to the O-RSfrom which it was derived, and furthermore, retains O-RS biologicalactivity; for example, a conservative variant O-RS that retains at least10% of the biological activity of the parent O-RS molecule from which itwas derived, or alternatively, at least 20%, at least 30%, or at least40%. In some preferred embodiments, the conservative variant O-RSretains at least 50% of the biological activity of the parent O-RSmolecule from which it was derived. The conservative amino acidsubstitutions of a conservative variant O-RS can occur in any domain ofthe O-RS, including the amino acid binding pocket.

Selection or screening agent: As used herein, the term “selection orscreening agent” refers to an agent that, when present, allows forselection/screening of certain components from a population. Forexample, a selection or screening agent can be, but is not limited to,e.g., a nutrient, an antibiotic, a wavelength of light, an antibody, anexpressed polynucleotide, or the like. The selection agent can bevaried, e.g., by concentration, intensity, etc.

In response to: As used herein, the term “in response to” refers to theprocess in which an O-tRNA of the invention recognizes a selector codonand mediates the incorporation of the unnatural amino acid, which iscoupled to the tRNA, into the growing polypeptide chain.

Encode: As used herein, the term “encode” refers to any process wherebythe information in a polymeric macromolecule or sequence string is usedto direct the production of a second molecule or sequence string that isdifferent from the first molecule or sequence string. As used herein,the term is used broadly, and can have a variety of applications. Insome aspects, the term “encode” describes the process ofsemi-conservative DNA replication, where one strand of a double-strandedDNA molecule is used as a template to encode a newly synthesizedcomplementary sister strand by a DNA-dependent DNA polymerase.

In another aspect, the term “encode” refers to any process whereby theinformation in one molecule is used to direct the production of a secondmolecule that has a different chemical nature from the first molecule.For example, a DNA molecule can encode an RNA molecule (e.g., by theprocess of transcription incorporating a DNA-dependent RNA polymeraseenzyme). Also, an RNA molecule can encode a polypeptide, as in theprocess of translation. When used to describe the process oftranslation, the term “encode” also extends to the triplet codon thatencodes an amino acid. In some aspects, an RNA molecule can encode a DNAmolecule, e.g., by the process of reverse transcription incorporating anRNA-dependent DNA polymerase. In another aspect, a DNA molecule canencode a polypeptide, where it is understood that “encode” as used inthat case incorporates both the processes of transcription andtranslation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the chemical structure of the unnatural amino acidsulfotyrosine.

FIG. 2 shows a denaturing PAGE gel stained with Coomassie blueillustrating sulfo-hirudin and sulfo-hirudin migration. Size of hirudincannot be judged by molecular weight standards due to hirudin's atypicalcharge.

FIGS. 3A and 3B provide representative plots of thrombin inhibition withtheir respective fitted progress curves superimposed on the raw datapoints. Enzyme assays were conducted with 50 μM fluorogenic substrate,40 pM human α-thrombin, and 100 pM expressed hirudin in a Tris-HClsaline buffer supplemented with polyethylene glycol 6000 and HSA. FIG.3A shows plots of fluorescence intensity over time for no inhibition(control), inhibition by desulfo-hirudin, and inhibition bysulfo-hirudin. FIG. 3B shows expansion of desulfo-hirudin andsulfo-hirudin plots for comparison.

FIGS. 4A and 4B illustrate sulfotyrosine dependent expression ofZ-domain. FIG. 4A provides a denaturing PAGE gel stained with Coomassieblue of Ni-NTA purified cell lysate from cells expressing Z-domain withan amber codon at position 7. Only expression with sulfotyrosinesupplemented media yields full-length Z-domain. FIG. 4B provides apositive-ion linear mode MALDI-TOF spectra (generated using THAP matrix)of Ni-NTA purified cell lysate (concentrated and dialyzed against water)showing a peak corresponding to full-length Z-domain containing a singlesulfotyrosine and lacking methionine. Also observed is a peakcorresponding to loss of sulfate resulting from mass spectral analysisconditions.

FIGS. 5A, 5B and 5C show various MALDI-TOF spectra. FIG. 5A shows apositive-ion linear mode MALDI-TOF spectra (generated using a THAPmatrix) of pure sulfo-hirudin showing both the intact [M+H]sulfo-hirudin peak (7059 Da) and the peak corresponding to loss ofsulfate during mass spectral analysis (6979 Da). Note that the smallpeaks to the right of the main ones are sodium adducts. They occur atadditional intervals of 22 Da. FIG. 5B shows a MALDI-TOF spectra(generated using a sinapinic matrix) documenting purity of the sample.To enhance detection of possible impurities, a harsher sinapinic matrix,which results in the predominance of the [M+H-80] peak, was used. Thepeak at 13964 Da can be attributed to dimerization of sulfo-hirudin. Noother impurities are observed. FIG. 5C shows an expansion of relevantregion to show presence of both [M+H-80] and intact sulfo-hirudin peak.Intact sulfo-hirudin is the minor peak due to the use of the harshersinapinic matrix. The small peaks to the right of the main ones aresodium adducts.

FIGS. 6A and 6B show various MALDI-TOF spectra. FIG. 6A shows aMALDI-TOF spectra (generated using a sinapinic matrix) of unpurifiedsulfo-hirudin expression media corresponding to expression in theabsence of sulfotyrosine. Only the truncated hirudin peak is found; nofull-length protein is observed. FIG. 6B shows a MALDI-TOF spectra(generated using a sinapinic matrix) of unpurified sulfo-hirudinexpression media corresponding to expression in the presence ofsulfotyrosine demonstrating the peak ratio of truncated to full-lengthsulfo-hirudin. Because of the harsher conditions necessary for gooddetection of crude sample mixtures, only the ionized form ofsulfo-hirudin is clearly observed.

FIG. 7 provides nucleotide and amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

Although tyrosine sulfation is a post-translational modificationwidespread across multicellular eukaryotes (Moore, “The biology andenzymology of protein tyrosine O-sulfation,” J Biol Chem 278:24243-24246(2003)), its biological functions remain largely unknown. This is inpart due to the difficulties associated with the synthesis ofselectively sulfated proteins. The invention provides for the selectiveincorporation of sulfotyrosine into proteins in bacteria by geneticallyencoding the modified amino acid in response to the amber nonsensecodon, TAG. Moreover, it is demonstrated that sulfo-hirudin, previouslyinaccessible through recombinant methods, can be directly expressed inE. coli using this strategy. As described herein, kinetic analyses showa greater than 10-fold enhancement in affinity towards human thrombin bysulfo-hirudin over desulfo-hirudin, an observation that offers clinicaladvantages for sulfo-hirudin in its use as an anticoagulant (Di Nisio etal., “Direct thrombin inhibitors,” N Engl J Med 353:1028-1040 (2005)).This general approach to the biosynthesis of sulfated proteinsfacilitates further study and application of the emergingpost-translational modification, tyrosine sulfation.

As a general method for the site-specific sulfation of proteins, thepresent describes the evolution of an orthogonal tRNA/aminoacyl-tRNAsynthetase (aaRS) pair that allows the efficient, selectiveincorporation of sulfotyrosine into proteins in eukaryotes such as E.coli in response to the amber nonsense codon. Using this uniquesuppressor tRNA/aaRS pair, the native sulfated form of hirudin isdirectly expressed and is shown that it has a greater than 10-foldhigher affinity for human thrombin than does desulfo-hirudin, inagreement with previous literature reports (Stone and Hofsteenge,“Kinetics of the inhibition of thrombin by hirudin,” Biochemistry25:4622-4628 (1986)).

The present specification provides orthogonal tRNA/aminoacyl-tRNAsynthetase pairs that allow the in vivo selective introduction ofsulfotyrosine (see FIG. 1) into proteins in E. coli in response to aselector codon, e.g., the amber stop codon TAG. The invention providesnovel orthogonal aminoacyl-tRNA synthetase (O-RS) polypeptides thatspecifically charge a cognate orthogonal tRNA (O-tRNA) with theunnatural amino acid sulfotyrosine.

In some aspects, to demonstrate (but not to limit) the presentinvention, the disclosure herein demonstrates that the unnatural aminoacid moiety can be incorporated into various model proteins. It is notintended that the incorporation of the unnatural amino acid be limitedto any particular protein. From the present disclosure, it will be clearthat the incorporation of the unnatural amino acid sulfotyrosine intoparticular proteins of interest is advantageous for a wide variety ofpurposes.

The present disclosure describes the evolution of novel orthogonaltRNA/aminoacyl-tRNA synthetase pairs that function in eubacteria to sitespecifically incorporate a sulfotyrosine unnatural amino acid (providedin FIG. 1) in response to selector codons. Briefly, the inventionprovides novel mutants of the Methanococcus janaschii tyrosyl-tRNAsynthetase that selectively charge a suppressor tRNA with the unnaturalamino acid sulfotyrosine in E. coli host cells.

These evolved tRNA-synthetase pairs can be used to site-specificallyincorporate the unnatural sulfotyrosine amino acid into a protein. Theincorporation of the unnatural amino acid into the protein can beprogrammed to occur at any desired position by engineering thepolynucleotide encoding the protein of interest to contain a selectorcodon that signals the incorporation of the unnatural amino acid.

The invention described herein provides orthogonal pairs for the geneticencoding and incorporation of the unnatural amino acid sulfotyrosineinto proteins in a eubacteria, e.g., an E. coli cell, where theorthogonal components do not cross-react with endogenous E. colicomponents of the translational machinery of the host cell, butrecognize the desired unnatural amino acid and incorporate it intoproteins in response to a selector codon (e.g., an amber nonsense codon,TAG). The orthogonal components provided by the invention includeorthogonal aminoacyl-tRNA synthetases derived from Methanococcusjannaschii tyrosyl tRNA-synthetase, and the mutant tyrosyl tRNA_(CUA)amber suppressor, which function as an orthogonal pair in a eubacterialhost cell.

This invention provides compositions of and methods for identifying andproducing additional orthogonal tRNA-aminoacyl-tRNA synthetase pairs,e.g., O-tRNA/O-RS pairs that can be used to incorporate sulfotyrosineinto proteins. An O-tRNA/O-RS pair of the invention is capable ofmediating incorporation of the sulfotyrosine into a protein that isencoded by a polynucleotide, where the polynucleotide comprises aselector codon that is recognized by the O-tRNA. The anticodon loop ofthe O-tRNA recognizes the selector codon on an mRNA and incorporates theunnatural amino acid at this site in the polypeptide. Generally, anorthogonal aminoacyl-tRNA synthetase of the invention preferentiallyaminoacylates (or charges) its O-tRNA with only one specific unnaturalamino acid.

Orthogonal tRNA/Aminoacyl-tRNA Synthetase Technology

An understanding of the novel compositions and methods of the presentinvention requires an understanding of the activities associated withorthogonal tRNA and orthogonal aminoacyl-tRNA synthetase pairs. In orderto add additional unnatural amino acids to the genetic code, neworthogonal pairs comprising an aminoacyl-tRNA synthetase and a suitabletRNA are needed that can function efficiently in the host translationalmachinery, but that are “orthogonal” to the translation system at issue,meaning that it functions independently of the synthetases and tRNAsendogenous to the translation system. Desired characteristics of theorthogonal pair include tRNA that decode or recognize only a specificcodon, e.g., a selector codon, that is not decoded by any endogenoustRNA, and aminoacyl-tRNA synthetases that preferentially aminoacylate(or “charge”) its cognate tRNA with only one specific unnatural aminoacid. The O-tRNA is also not typically aminoacylated (or is poorlyaminoacylated, i.e., charged) by endogenous synthetases. For example, inan E. coli host system, an orthogonal pair will include anaminoacyl-tRNA synthetase that does not cross-react with any of theendogenous tRNA, e.g., which there are 40 in E. coli, and an orthogonaltRNA that is not aminoacylated by any of the endogenous synthetases,e.g., of which there are 21 in E. coli.

The general principles of orthogonal translation systems that aresuitable for making proteins that comprise one or more unnatural aminoacid are known in the art, as are the general methods for producingorthogonal translation systems. For example, see InternationalPublication Numbers WO 2002/086075, entitled “METHODS AND COMPOSITIONFOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;”WO 2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINOACIDS;” WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC GENETICCODE;” WO 2005/019415, filed Jul. 7, 2004; WO 2005/007870, filed Jul. 7,2004; WO 2005/007624, filed Jul. 7, 2004; WO 2006/110182, filed Oct. 27,2005, entitled “ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVOINCORPORATION OF UNNATURAL AMINO ACIDS” and WO 2007/103490, filed Mar.7, 2007, entitled “SYSTEMS FOR THE EXPRESSION OF ORTHOGONAL TRANSLATIONCOMPONENTS IN EUBACTERIAL HOST CELLS.” Each of these applications isincorporated herein by reference in its entirety. For discussion oforthogonal translation systems that incorporate unnatural amino acids,and methods for their production and use, see also, Wang and Schultz“Expanding the Genetic Code,” Angewandte Chemie Int. Ed., 44(1):34-66(2005), Xie and Schultz, “An Expanding Genetic Code,” Methods36(3):227-238 (2005); Xie and Schultz, “Adding Amino Acids to theGenetic Repertoire,” Curr. Opinion in Chemical Biology 9(6):548-554(2005); and Wang et al., “Expanding the Genetic Code,” Annu. Rev.Biophys. Biomol. Struct., 35:225-249 (2006); the contents of which areeach incorporated by reference in their entirety.

Orthogonal Translation Systems

Orthogonal translation systems generally comprise cells (which can beprokaryotic cells such as E. coli) that include an orthogonal tRNA(O-tRNA), an orthogonal aminoacyl tRNA synthetase (O-RS), and anunnatural amino acid, where the O-RS aminoacylates the O-tRNA with theunnatural amino acid. An orthogonal pair of the invention can include anO-tRNA, e.g., a suppressor tRNA, a frameshift tRNA, or the like, and acognate O-RS. The orthogonal systems of the invention can typicallycomprise O-tRNA/O-RS pairs, either in the context of a host cell orwithout the host cell. In addition to multi-component systems, theinvention also provides novel individual components, for example, novelorthogonal aminoacyl-tRNA synthetase polypeptides (e.g., SEQ ID NO: 4,6, 8 or 10), and the polynucleotides that encodes those polypeptides(e.g., SEQ ID NO: 5, 7, 9 or 11).

In general, when an orthogonal pair recognizes a selector codon andloads an amino acid in response to the selector codon, the orthogonalpair is said to “suppress” the selector codon. That is, a selector codonthat is not recognized by the translation system's (e.g., the cell's)endogenous machinery is not ordinarily charged, which results inblocking production of a polypeptide that would otherwise be translatedfrom the nucleic acid. In an orthogonal pair system, the O-RSaminoacylates the O-tRNA with a specific unnatural amino acid. Thecharged O-tRNA recognizes the selector codon and suppresses thetranslational block caused by the selector codon.

In some aspects, an O-tRNA of the invention recognizes a selector codonand includes at least about, e.g., a 45%, a 50%, a 60%, a 75%, a 80%, ora 90% or more suppression efficiency in the presence of a cognatesynthetase in response to a selector codon as compared to thesuppression efficiency of an O-tRNA comprising or encoded by apolynucleotide sequence as set forth in the sequence listing herein.

In some embodiments, the suppression efficiency of the O-RS and theO-tRNA together is about, e.g., 5 fold, 10 fold, 15 fold, 20 fold, or 25fold or more greater than the suppression efficiency of the O-tRNAlacking the O-RS. In some aspect, the suppression efficiency of the O-RSand the O-tRNA together is at least about, e.g., 35%, 40%, 45%, 50%,60%, 75%, 80%, or 90% or more of the suppression efficiency of anorthogonal synthetase pair as set forth in the sequence listings herein.

The host cell uses the O-tRNA/O-RS pair to incorporate the unnaturalamino acid into a growing polypeptide chain, e.g., via a nucleic acidthat comprises a polynucleotide that encodes a polypeptide of interest,where the polynucleotide comprises a selector codon that is recognizedby the O-tRNA. In certain preferred aspects, the cell can include one ormore additional O-tRNA/O-RS pairs, where the additional O-tRNA is loadedby the additional O-RS with a different unnatural amino acid. Forexample, one of the O-tRNAs can recognize a four base codon and theother O-tRNA can recognize a stop codon. Alternately, multiple differentstop codons or multiple different four base codons can be used in thesame coding nucleic acid.

As noted, in some embodiments, there exists multiple O-tRNA/O-RS pairsin a cell or other translation system, which allows incorporation ofmore than one unnatural amino acid into a polypeptide. For example, thecell can further include an additional different O-tRNA/O-RS pair and asecond unnatural amino acid, where this additional O-tRNA recognizes asecond selector codon and this additional O-RS preferentiallyaminoacylates the O-tRNA with the second unnatural amino acid. Forexample, a cell that includes an O-tRNA/O-RS pair (where the O-tRNArecognizes, e.g., an amber selector codon), can further comprise asecond orthogonal pair, where the second O-tRNA recognizes a differentselector codon, e.g., an opal codon, a four-base codon, or the like.Desirably, the different orthogonal pairs are derived from differentsources, which can facilitate recognition of different selector codons.

In certain embodiments, systems comprise a cell such as an E. coli cellthat includes an orthogonal tRNA (O-tRNA), an orthogonal aminoacyl-tRNAsynthetase (O-RS), an unnatural amino acid and a nucleic acid thatcomprises a polynucleotide that encodes a polypeptide of interest, wherethe polynucleotide comprises the selector codon that is recognized bythe O-tRNA. The translation system can also be a cell-free system, e.g.,any of a variety of commercially available “in vitro”transcription/translation systems in combination with an O-tRNA/O-RSpair and an unnatural amino acid as described herein.

The O-tRNA and/or the O-RS can be naturally occurring or can be, e.g.,derived by mutation of a naturally occurring tRNA and/or RS, e.g., bygenerating libraries of tRNAs and/or libraries of RSs, from any of avariety of organisms and/or by using any of a variety of availablemutation strategies. For example, one strategy for producing anorthogonal tRNA/aminoacyl-tRNA synthetase pair involves importing aheterologous (to the host cell) tRNA/synthetase pair from, e.g., asource other than the host cell, or multiple sources, into the hostcell. The properties of the heterologous synthetase candidate include,e.g., that it does not charge any host cell tRNA, and the properties ofthe heterologous tRNA candidate include, e.g., that it is notaminoacylated by any host cell synthetase. In addition, the heterologoustRNA is orthogonal to all host cell synthetases. A second strategy forgenerating an orthogonal pair involves generating mutant libraries fromwhich to screen and/or select an O-tRNA or O-RS. These strategies canalso be combined.

Orthogonal tRNA (O-tRNA)

An orthogonal tRNA (O-tRNA) of the invention desirably mediatesincorporation of an unnatural amino acid into a protein that is encodedby a polynucleotide that comprises a selector codon that is recognizedby the O-tRNA, e.g., in vivo or in vitro. In certain embodiments, anO-tRNA of the invention includes at least about, e.g., a 45%, a 50%, a60%, a 75%, a 80%, or a 90% or more suppression efficiency in thepresence of a cognate synthetase in response to a selector codon ascompared to an O-tRNA comprising or encoded by a polynucleotide sequenceas set forth in the O-tRNA sequences in the sequence listing herein.

Suppression efficiency can be determined by any of a number of assaysknown in the art. For example, a β-galactosidase reporter assay can beused, e.g., a derivatized lacZ plasmid (where the construct has aselector codon n the lacZ nucleic acid sequence) is introduced intocells from an appropriate organism (e.g., an organism where theorthogonal components can be used) along with plasmid comprising anO-tRNA of the invention. A cognate synthetase can also be introduced(either as a polypeptide or a polynucleotide that encodes the cognatesynthetase when expressed). The cells are grown in media to a desireddensity, e.g., to an OD₆₀₀ of about 0.5, and β-galactosidase assays areperformed, e.g., using the BetaFluor™ β-Galactosidase Assay Kit(Novagen). Percent suppression can be calculated as the percentage ofactivity for a sample relative to a comparable control, e.g., the valueobserved from the derivatized lacZ construct, where the construct has acorresponding sense codon at desired position rather than a selectorcodon.

Examples of O-tRNAs of the invention are set forth in the sequencelisting herein, for example, see FIG. 7 and SEQ ID NO: 1. The disclosureherein also provides guidance for the design of additional equivalentO-tRNA species. In an RNA molecule, such as an O-RS mRNA, or O-tRNAmolecule, Thymine (T) is replace with Uracil (U) relative to a givensequence (or vice versa for a coding DNA), or complement thereof.Additional modifications to the bases can also be present to generatelargely functionally equivalent molecules.

The invention also encompasses conservative variations of O-tRNAscorresponding to particular O-tRNAs herein. For example, conservativevariations of O-tRNA include those molecules that function like theparticular O-tRNAs, e.g., as in the sequence listing herein and thatmaintain the tRNA L-shaped structure by virtue of appropriateself-complementarity, but that do not have a sequence identical tothose, e.g., in the sequence listing or FIG. 7, and desirably, are otherthan wild type tRNA molecules.

The composition comprising an O-tRNA can further include an orthogonalaminoacyl-tRNA synthetase (O-RS), where the O-RS preferentiallyaminoacylates the O-tRNA with an unnatural amino acid. In certainembodiments, a composition including an O-tRNA can further include atranslation system (e.g., in vitro or in vivo). A nucleic acid thatcomprises a polynucleotide that encodes a polypeptide of interest, wherethe polynucleotide comprises a selector codon that is recognized by theO-tRNA, or a combination of one or more of these can also be present inthe cell.

Methods of producing an orthogonal tRNA (O-tRNA) are also a feature ofthe invention. An O-tRNA produced by the method is also a feature of theinvention. In certain embodiments of the invention, the O-tRNAs can beproduced by generating a library of mutants. The library of mutant tRNAscan be generated using various mutagenesis techniques known in the art.For example, the mutant tRNAs can be generated by site-specificmutations, random point mutations, homologous recombination, DNAshuffling or other recursive mutagenesis methods, chimeric constructionor any combination thereof, e.g., of the O-tRNA of SEQ ID NO: 1.

Additional mutations can be introduced at a specific position(s), e.g.,at a nonconservative position(s), or at a conservative position, at arandomized position(s), or a combination of both in a desired loop orregion of a tRNA, e.g., an anticodon loop, the acceptor stem, D arm orloop, variable loop, TPC arm or loop, other regions of the tRNAmolecule, or a combination thereof. Typically, mutations in a tRNAinclude mutating the anticodon loop of each member of the library ofmutant tRNAs to allow recognition of a selector codon. The method canfurther include adding additional sequences to the O-tRNA. Typically, anO-tRNA possesses an improvement of orthogonality for a desired organismcompared to the starting material, e.g., the plurality of tRNAsequences, while preserving its affinity towards a desired RS.

The methods optionally include analyzing the similarity (and/or inferredhomology) of sequences of tRNAs and/or aminoacyl-tRNA synthetases todetermine potential candidates for an O-tRNA, O-RS and/or pairs thereof,that appear to be orthogonal for a specific organism. Computer programsknown in the art and described herein can be used for the analysis,e.g., BLAST and pileup programs can be used. In one example, to choosepotential orthogonal translational components for use in E. coli, asynthetase and/or a tRNA is chosen that does not display close sequencesimilarity to eubacterial organisms.

Typically, an O-tRNA is obtained by subjecting to, e.g., negativeselection, a population of cells of a first species, where the cellscomprise a member of the plurality of potential O-tRNAs. The negativeselection eliminates cells that comprise a member of the library ofpotential O-tRNAs that is aminoacylated by an aminoacyl-tRNA synthetase(RS) that is endogenous to the cell. This provides a pool of tRNAs thatare orthogonal to the cell of the first species.

In certain embodiments, in the negative selection, a selector codon(s)is introduced into a polynucleotide that encodes a negative selectionmarker, e.g., an enzyme that confers antibiotic resistance, e.g.,β-lactamase, an enzyme that confers a detectable product, e.g.,β-galactosidase, chloramphenicol acetyltransferase (CAT), e.g., a toxicproduct, such as barnase, at a nonessential position (e.g., stillproducing a functional barnase), etc. Screening/selection is optionallydone by growing the population of cells in the presence of a selectiveagent (e.g., an antibiotic, such as ampicillin). In one embodiment, theconcentration of the selection agent is varied.

For example, to measure the activity of suppressor tRNAs, a selectionsystem is used that is based on the in vivo suppression of selectorcodon, e.g., nonsense (e.g., stop) or frameshift mutations introducedinto a polynucleotide that encodes a negative selection marker, e.g., agene for β-lactamase (bla). For example, polynucleotide variants, e.g.,bla variants, with a selector codon at a certain position (e.g., A184),are constructed. Cells, e.g., bacteria, are transformed with thesepolynucleotides. In the case of an orthogonal tRNA, which cannot beefficiently charged by endogenous E. coli synthetases, antibioticresistance, e.g., ampicillin resistance, should be about or less thanthat for a bacteria transformed with no plasmid. If the tRNA is notorthogonal, or if a heterologous synthetase capable of charging the tRNAis co-expressed in the system, a higher level of antibiotic, e.g.,ampicillin, resistance is be observed. Cells, e.g., bacteria, are chosenthat are unable to grow on LB agar plates with antibiotic concentrationsabout equal to cells transformed with no plasmids.

In the case of a toxic product (e.g., ribonuclease or barnase), when amember of the plurality of potential tRNAs is aminoacylated byendogenous host, e.g., Escherichia coli synthetases (i.e., it is notorthogonal to the host, e.g., Escherichia coli synthetases), theselector codon is suppressed and the toxic polynucleotide productproduced leads to cell death. Cells harboring orthogonal tRNAs ornon-functional tRNAs survive.

In one embodiment, the pool of tRNAs that are orthogonal to a desiredorganism are then subjected to a positive selection in which a selectorcodon is placed in a positive selection marker, e.g., encoded by a drugresistance gene, such a β-lactamase gene. The positive selection isperformed on a cell comprising a polynucleotide encoding or comprising amember of the pool of tRNAs that are orthogonal to the cell, apolynucleotide encoding a positive selection marker, and apolynucleotide encoding a cognate RS. In certain embodiments, the secondpopulation of cells comprises cells that were not eliminated by thenegative selection. The polynucleotides are expressed in the cell andthe cell is grown in the presence of a selection agent, e.g.,ampicillin. tRNAs are then selected for their ability to beaminoacylated by the coexpressed cognate synthetase and to insert anamino acid in response to this selector codon. Typically, these cellsshow an enhancement in suppression efficiency compared to cellsharboring non-functional tRNA(s), or tRNAs that cannot efficiently berecognized by the synthetase of interest. The cell harboring thenon-functional tRNAs or tRNAs that are not efficiently recognized by thesynthetase of interest, are sensitive to the antibiotic. Therefore,tRNAs that: (i) are not substrates for endogenous host, e.g.,Escherichia coli, synthetases; (ii) can be aminoacylated by thesynthetase of interest; and (iii) are functional in translation, surviveboth selections.

Accordingly, the same marker can be either a positive or negativemarker, depending on the context in which it is screened. That is, themarker is a positive marker if it is screened for, but a negative markerif screened against.

The stringency of the selection, e.g., the positive selection, thenegative selection or both the positive and negative selection, in theabove described-methods, optionally includes varying the selectionstringency. For example, because barnase is an extremely toxic protein,the stringency of the negative selection can be controlled byintroducing different numbers of selector codons into the barnase geneand/or by using an inducible promoter. In another example, theconcentration of the selection or screening agent is varied (e.g.,ampicillin concentration). In some aspects of the invention, thestringency is varied because the desired activity can be low duringearly rounds. Thus, less stringent selection criteria are applied inearly rounds and more stringent criteria are applied in later rounds ofselection. In certain embodiments, the negative selection, the positiveselection or both the negative and positive selection can be repeatedmultiple times. Multiple different negative selection markers, positiveselection markers or both negative and positive selection markers can beused. In certain embodiments, the positive and negative selection markercan be the same.

Other types of selections/screening can be used in the invention forproducing orthogonal translational components, e.g., an O-tRNA, an O-RS,and an O-tRNA/O-RS pair that loads an unnatural amino acid in responseto a selector codon. For example, the negative selection marker, thepositive selection marker or both the positive and negative selectionmarkers can include a marker that fluoresces or catalyzes a luminescentreaction in the presence of a suitable reactant. In another embodiment,a product of the marker is detected by fluorescence-activated cellsorting (FACS) or by luminescence. Optionally, the marker includes anaffinity based screening marker. See also, Francisco, J. A., et al.,(1993) Production and fluorescence-activated cell sorting of Escherichiacoli expressing a functional antibody fragment on the external surface.Proc Natl Acad Sci USA. 90:10444-8.

Additional methods for producing a recombinant orthogonal tRNA can befound, e.g., in International Application Publications WO 2002/086075,entitled “METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNAAMINOACYL-tRNA SYNTHETASE PAIRS;” WO 2004/094593, entitled “EXPANDINGTHE EUKARYOTIC GENETIC CODE;” and WO 2005/019415, filed Jul. 7, 2004.See also Forster et al., (2003) Programming peptidomimetic synthetasesby translating genetic codes designed de novo PNAS 100(11):6353-6357;and, Feng et al., (2003), Expanding tRNA recognition of a tRNAsynthetase by a single amino acid change, PNAS 100(10): 5676-5681.

Orthogonal Aminoacyl-tRNA Synthetase (O-RS)

An O-RS of the invention preferentially aminoacylates an O-tRNA with anunnatural amino acid, in vitro or in vivo. An O-RS of the invention canbe provided to the translation system, e.g., a cell, by a polypeptidethat includes an O-RS and/or by a polynucleotide that encodes an O-RS ora portion thereof. For example, an example O-RS comprises an amino acidsequence as set forth in SEQ ID NO: 4, 6, 8 or 10, or a conservativevariation thereof. In another example, an O-RS, or a portion thereof, isencoded by a polynucleotide sequence that encodes an amino acidcomprising sequence in the sequence listing or examples herein, or acomplementary polynucleotide sequence thereof. See, e.g., thepolynucleotide of SEQ ID NO: 5, 7, 9 or 11.

Methods for identifying an orthogonal aminoacyl-tRNA synthetase (O-RS),e.g., an O-RS, for use with an O-tRNA, are also a feature of theinvention. For example, a method includes subjecting to selection, e.g.,positive selection, a population of cells of a first species, where thecells individually comprise: 1) a member of a plurality ofaminoacyl-tRNA synthetases (RSs), (e.g., the plurality of RSs caninclude mutant RSs, RSs derived from a species other than the firstspecies or both mutant RSs and RSs derived from a species other than thefirst species); 2) the orthogonal tRNA (O-tRNA) (e.g., from one or morespecies); and 3) a polynucleotide that encodes an (e.g., positive)selection marker and comprises at least one selector codon. Cells areselected or screened for those that show an enhancement in suppressionefficiency compared to cells lacking or with a reduced amount of themember of the plurality of RSs. Suppression efficiency can be measuredby techniques known in the art and as described herein. Cells having anenhancement in suppression efficiency comprise an active RS thataminoacylates the O-tRNA. A level of aminoacylation (in vitro or invivo) by the active RS of a first set of tRNAs from the first species iscompared to the level of aminoacylation (in vitro or in vivo) by theactive RS of a second set of tRNAs from the second species. The level ofaminoacylation can be determined by a detectable substance (e.g., alabeled unnatural amino acid). The active RS that more efficientlyaminoacylates the second set of tRNAs compared to the first set of tRNAsis typically selected, thereby providing an efficient (optimized)orthogonal aminoacyl-tRNA synthetase for use with the O-tRNA. An O-RS,identified by the method, is also a feature of the invention.

Any of a number of assays can be used to determine aminoacylation. Theseassays can be performed in vitro or in vivo. For example, in vitroaminoacylation assays are described in, e.g., Hoben and Soll (1985)Methods Enzymol. 113:55-59. Aminoacylation can also be determined byusing a reporter along with orthogonal translation components anddetecting the reporter in a cell expressing a polynucleotide comprisingat least one selector codon that encodes a protein. See also, WO2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;”and WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC GENETIC CODE.”

Identified O-RS can be further manipulated to alter substratespecificity of the synthetase, so that only a desired unnatural aminoacid, but not any of the common 20 amino acids, are charged to theO-tRNA. Methods to generate an orthogonal aminoacyl-tRNA synthetase witha substrate specificity for an unnatural amino acid include mutating thesynthetase, e.g., at the active site in the synthetase, at the editingmechanism site in the synthetase, at different sites by combiningdifferent domains of synthetases, or the like, and applying a selectionprocess. A strategy is used, which is based on the combination of apositive selection followed by a negative selection. In the positiveselection, suppression of the selector codon introduced at anonessential position(s) of a positive marker allows cells to surviveunder positive selection pressure. In the presence of both natural andunnatural amino acids, survivors thus encode active synthetases chargingthe orthogonal suppressor tRNA with either a natural or unnatural aminoacid. In the negative selection, suppression of a selector codonintroduced at a nonessential position(s) of a negative marker removessynthetases with natural amino acid specificities. Survivors of thenegative and positive selection encode synthetases that aminoacylate(charge) the orthogonal suppressor tRNA with unnatural amino acids only.These synthetases can then be subjected to further mutagenesis, e.g.,DNA shuffling or other recursive mutagenesis methods.

A library of mutant O-RSs can be generated using various mutagenesistechniques known in the art. For example, the mutant RSs can begenerated by site-specific mutations, random point mutations, homologousrecombination, DNA shuffling or other recursive mutagenesis methods,chimeric construction or any combination thereof. For example, a libraryof mutant RSs can be produced from two or more other, e.g., smaller,less diverse “sub-libraries.” Chimeric libraries of RSs are alsoincluded in the invention. It should be noted that libraries of tRNAsynthetases from various organism (e.g., microorganisms such aseubacteria or archaebacteria) such as libraries that comprise naturaldiversity (see, e.g., U.S. Pat. No. 6,238,884 to Short et al.; U.S. Pat.No. 5,756,316 to Schallenberger et al.; U.S. Pat. No. 5,783,431 toPetersen et al.; U.S. Pat. No. 5,824,485 to Thompson et al.; U.S. Pat.No. 5,958,672 to Short et al.), are optionally constructed and screenedfor orthogonal pairs.

Once the synthetases are subject to the positive and negativeselection/screening strategy, these synthetases can then be subjected tofurther mutagenesis. For example, a nucleic acid that encodes the O-RScan be isolated; a set of polynucleotides that encode mutated O-RSs(e.g., by random mutagenesis, site-specific mutagenesis, recombinationor any combination thereof) can be generated from the nucleic acid; and,these individual steps or a combination of these steps can be repeateduntil a mutated O-RS is obtained that preferentially aminoacylates theO-tRNA with the unnatural amino acid. In some aspects of the invention,the steps are performed multiple times, e.g., at least two times.

Additional levels of selection/screening stringency can also be used inthe methods of the invention, for producing O-tRNA, O-RS, or pairsthereof. The selection or screening stringency can be varied on one orboth steps of the method to produce an O-RS. This could include, e.g.,varying the amount of selection/screening agent that is used, etc.Additional rounds of positive and/or negative selections can also beperformed. Selecting or screening can also comprise one or more of achange in amino acid permeability, a change in translation efficiency, achange in translational fidelity, etc. Typically, the one or more changeis based upon a mutation in one or more gene in an organism in which anorthogonal tRNA-tRNA synthetase pair is used to produce protein.

Additional general details for producing O-RS, and altering thesubstrate specificity of the synthetase can be found in InternalPublication Number WO 2002/086075, entitled “METHODS AND COMPOSITIONSFOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS;”and WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC GENETIC CODE.”See also, Wang and Schultz “Expanding the Genetic Code,” AngewandteChemie Int. Ed., 44(1):34-66 (2005), the content of which isincorporated by reference in its entirety.

Source and Host Organisms

The orthogonal translational components (O-tRNA and O-RS) of theinvention can be derived from any organism (or a combination oforganisms) for use in a host translation system from any other species,with the caveat that the O-tRNA/O-RS components and the host system workin an orthogonal manner. It is not a requirement that the O-tRNA and theO-RS from an orthogonal pair be derived from the same organism. In someaspects, the orthogonal components are derived from Archaea genes (i.e.,archaebacteria) for use in a eubacterial host system.

For example, the orthogonal O-tRNA can be derived from an Archaeorganism, e.g., an archaebacterium, such as Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix,Methanococcus maripaludis, Methanopyrus kandleri, Methanosarcina mazei(Mm), Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus(Ss), Sulfolobus tokodaii, Thermoplasma acidophilum, Thermoplasmavolcanium, or the like, or a eubacterium, such as Escherichia coli,Thermus thermophilus, Bacillus subtilis, Bacillus stearothermphilus, orthe like, while the orthogonal O-RS can be derived from an organism orcombination of organisms, e.g., an archaebacterium, such asMethanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium such as Haloferax volcanii and Halobacterium speciesNRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcushorikoshii, Aeuropyrum pernix, Methanococcus maripaludis, Methanopyruskandleri, Methanosarcina mazei, Pyrobaculum aerophilum, Pyrococcusabyssi, Sulfolobus solfataricus, Sulfolobus tokodaii, Thermoplasmaacidophilum, Thermoplasma volcanium, or the like, or a eubacterium, suchas Escherichia coli, Thermus thermophilus, Bacillus subtilis, Bacillusstearothermphilus, or the like. In one embodiment, eukaryotic sources,e.g., plants, algae, protists, fungi, yeasts, animals (e.g., mammals,insects, arthropods, etc.), or the like, can also be used as sources ofO-tRNAs and O-RSs.

The individual components of an O-tRNA/O-RS pair can be derived from thesame organism or different organisms. In one embodiment, the O-tRNA/O-RSpair is from the same organism. Alternatively, the O-tRNA and the O-RSof the O-tRNA/O-RS pair are from different organisms.

The O-tRNA, O-RS or O-tRNA/O-RS pair can be selected or screened in vivoor in vitro and/or used in a cell, e.g., a eubacterial cell, to producea polypeptide with an unnatural amino acid. The eubacterial cell used isnot limited, for example, Escherichia coli, Thermus thermophilus,Bacillus subtilis, Bacillus stearothermphilus, or the like. Compositionsof eubacterial cells comprising translational components of theinvention are also a feature of the invention.

See also, International Application Publication Number WO 2004/094593,entitled “EXPANDING THE EUKARYOTIC GENETIC CODE,” filed Apr. 16, 2004,for screening O-tRNA and/or O-RS in one species for use in anotherspecies.

Although orthogonal translation systems (e.g., comprising an O-RS, anO-tRNA and an unnatural amino acid) can utilize cultured host cells toproduce proteins having unnatural amino acids, it is not intended thatan orthogonal translation system of the invention require an intact,viable host cell. For example, a orthogonal translation system canutilize a cell-free system in the presence of a cell extract. Indeed,the use of cell free, in vitro transcription/translation systems forprotein production is a well established technique. Adaptation of thesein vitro systems to produce proteins having unnatural amino acids usingorthogonal translation system components described herein is well withinthe scope of the invention.

Selector Codons

Selector codons of the invention expand the genetic codon framework ofprotein biosynthetic machinery. For example, a selector codon includes,e.g., a unique three base codon, a nonsense codon, such as a stop codon,e.g., an amber codon (UAG), or an opal codon (UGA), an unnatural codon,at least a four base codon, a rare codon, or the like. A number ofselector codons can be introduced into a desired gene, e.g., one ormore, two or more, more than three, etc. By using different selectorcodons, multiple orthogonal tRNA/synthetase pairs can be used that allowthe simultaneous site-specific incorporation of multiple unnatural aminoacids e.g., including at least one unnatural amino acid, using thesedifferent selector codons.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of an unnatural amino acid in vivoin a cell. For example, an O-tRNA is produced that recognizes the stopcodon and is aminoacylated by an O-RS with an unnatural amino acid. ThisO-tRNA is not recognized by the naturally occurring host'saminoacyl-tRNA synthetases. Conventional site-directed mutagenesis canbe used to introduce the stop codon at the site of interest in apolynucleotide encoding a polypeptide of interest. See, e.g., Sayers etal. (1988), 5′,3′ Exonuclease in phosphorothioate-basedoligonucleotide-directed mutagenesis. Nucleic Acids Res, 791-802. Whenthe O-RS, O-tRNA and the nucleic acid that encodes a polypeptide ofinterest are combined, e.g., in vivo, the unnatural amino acid isincorporated in response to the stop codon to give a polypeptidecontaining the unnatural amino acid at the specified position. In oneembodiment of the invention, the stop codon used as a selector codon isan amber codon, UAG, and/or an opal codon, UGA. In one example, agenetic code in which UAG and UGA are both used as a selector codon canencode 22 amino acids while preserving the ochre nonsense codon, UAA,which is the most abundant termination signal.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the host cell. For example in non-eukaryoticcells, such as Escherichia coli, because the suppression efficiency forthe UAG codon depends upon the competition between the O-tRNA, e.g., theamber suppressor tRNA, and the release factor 1 (RF1) (which binds tothe UAG codon and initiates release of the growing peptide from theribosome), the suppression efficiency can be modulated by, e.g., eitherincreasing the expression level of O-tRNA, e.g., the suppressor tRNA, orusing an RF1 deficient strain. In eukaryotic cells, because thesuppression efficiency for the UAG codon depends upon the competitionbetween the O-tRNA, e.g., the amber suppressor tRNA, and a eukaryoticrelease factor (e.g., eRF) (which binds to a stop codon and initiatesrelease of the growing peptide from the ribosome), the suppressionefficiency can be modulated by, e.g., increasing the expression level ofO-tRNA, e.g., the suppressor tRNA. In addition, additional compounds canalso be present, e.g., reducing agents such as dithiothretiol (DTT).

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In thiscase, the synthetic tRNA competes with the naturally occurringtRNA^(Arg), which exists as a minor species in Escherichia coli. Inaddition, some organisms do not use all triplet codons. An unassignedcodon AGA in Micrococcus luteus has been utilized for insertion of aminoacids in an in vitro transcription/translation extract. See, e.g., Kowaland Oliver, Nucl. Acid. Res., 25:4685 (1997). Components of theinvention can be generated to use these rare codons in vivo.

Selector codons can also comprise extended codons, e.g., four or morebase codons, such as, four, five, six or more base codons. Examples offour base codons include, e.g., AGGA, CUAG, UAGA, CCCU, and the like.Examples of five base codons include, e.g., AGGAC, CCCCU, CCCUC, CUAGA,CUACU, UAGGC and the like. Methods of the invention include usingextended codons based on frameshift suppression. Four or more basecodons can insert, e.g., one or multiple unnatural amino acids, into thesame protein. In other embodiments, the anticodon loops can decode,e.g., at least a four-base codon, at least a five-base codon, or atleast a six-base codon or more. Since there are 256 possible four-basecodons, multiple unnatural amino acids can be encoded in the same cellusing a four or more base codon. See also, Anderson et al., (2002)“Exploring the Limits of Codon and Anticodon Size,” Chemistry andBiology 9:237-244; and, Magliery, (2001) “Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli,” J. Mol. Biol., 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry 32:7939; and Hohsaka et al., (1999)J. Am. Chem. Soc., 121:34. CGGG and AGGU were used to simultaneouslyincorporate 2-naphthylalanine and an NBD derivative of lysine intostreptavidin in vitro with two chemically acylated frameshift suppressortRNAs. See, e.g., Hohsaka et al., (1999) J. Am. Chem. Soc., 121:12194.In an in vivo study, Moore et al. examined the ability of tRNA^(Leu)derivatives with NCUA anticodons to suppress UAGN codons (N can be U, A,G, or C), and found that the quadruplet UAGA can be decoded by atRNA^(Leu) with a UCUA anticodon with an efficiency of 13 to 26% withlittle decoding in the 0 or −1 frame. See Moore et al., (2000) J. Mol.Biol., 298:195. In one embodiment, extended codons based on rare codonsor nonsense codons can be used in invention, which can reduce missensereadthrough and frameshift suppression at other unwanted sites. Fourbase codons have been used as selector codons in a variety of orthogonalsystems. See, e.g., WO 2005/019415; WO 2005/007870 and WO 2005/07624.See also, Wang and Schultz “Expanding the Genetic Code,” AngewandteChemie Int. Ed., 44(1):34-66 (2005), the content of which isincorporated by reference in its entirety. While the examples belowutilize an amber selector codon, four or more base codons can be used aswell, by modifying the examples herein to include four-base O-tRNAs andsynthetases modified to include mutations similar to those previouslydescribed for various unnatural amino acid O-RSs.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) “An unnatural base pair for incorporating amino acid analoguesinto protein,” Nature Biotechnology 20:177-182. See also Wu et al.,(2002) J. Am. Chem. Soc., 124:14626-14630. Other relevant publicationsare listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11586; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274. A3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., Tae et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is inserted into a gene but is nottranslated into protein. The sequence contains a structure that servesas a cue to induce the ribosome to hop over the sequence and resumetranslation downstream of the insertion.

Unnatural Amino Acids

As used herein, an unnatural amino acid refers to any amino acid,modified amino acid, or amino acid analogue other than selenocysteineand/or pyrrolysine and the following twenty genetically encodedalpha-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine. The generic structure of an alpha-aminoacid is illustrated by Formula I:

An unnatural amino acid is typically any structure having Formula Iwherein the R group is any substituent other than one used in the twentynatural amino acids. See e.g., Biochemistry by L. Stryer, 3^(rd) ed.1988, Freeman and Company, New York, for structures of the twentynatural amino acids. Note that, the unnatural amino acids of theinvention can be naturally occurring compounds other than the twentyalpha-amino acids above.

Because the unnatural amino acids of the invention typically differ fromthe natural amino acids in side chain, the unnatural amino acids formamide bonds with other amino acids, e.g., natural or unnatural, in thesame manner in which they are formed in naturally occurring proteins.However, the unnatural amino acids have side chain groups thatdistinguish them from the natural amino acids.

Of particular interest herein is the unnatural amino acid sulfotyrosine(see FIG. 1). In addition to the sulfotyrosine unnatural amino acid,other unnatural amino acids can be simultaneously incorporated into apolypeptide of interest, e.g., using an appropriate second O-RS/O-tRNApair in conjunction with an orthogonal pair provided by the presentinvention. Many such additional unnatural amino acids and suitableorthogonal pairs are known. See the present disclosure and thereferences cited herein. For example, see Wang and Schultz “Expandingthe Genetic Code,” Angewandte Chemie Int. Ed., 44(1):34-66 (2005); Xieand Schultz, “An Expanding Genetic Code,” Methods 36(3):227-238 (2005);Xie and Schultz, “Adding Amino Acids to the Genetic Repertoire,” Curr.Opinion in Chemical Biology 9(6):548-554 (2005); and Wang et al.,“Expanding the Genetic Code,” Annu. Rev. Biophys. Biomol. Struct.,35:225-249 (2006); the contents of which are each incorporated byreference in their entirety.

Although the sulfotyrosine unnatural amino acid shown in FIG. 1 is ofprimary interest in the Examples described herein, it is not intendedthat the invention be strictly limited to that structure. Indeed, avariety of easily-derived, structurally related analogs can be readilyproduced that retain the principle characteristic of the sulfotyrosineshown in FIG. 1, and also are specifically recognized by theaminoacyl-tRNA synthetases of the invention (e.g., the O-RS of SEQ IDNOS: 4, 6, 8 and 10). It is intended that these related amino acidanalogues are within the scope of the invention.

In other unnatural amino acids, for example, R in Formula I optionallycomprises an alkyl-, aryl-, acyl-, hydrazine, cyano-, halo-, hydrazide,alkenyl, ether, borate, boronate, phospho, phosphono, phosphine, enone,imine, ester, hydroxylamine, amine, and the like, or any combinationthereof. Other unnatural amino acids of interest include, but are notlimited to, amino acids comprising a photoactivatable cross-linker,spin-labeled amino acids, fluorescent amino acids, metal binding aminoacids, metal-containing amino acids, radioactive amino acids, aminoacids with novel functional groups, amino acids that covalently ornoncovalently interact with other molecules, photocaged and/orphotoisomerizable amino acids, biotin or biotin-analogue containingamino acids, keto containing amino acids, glycosylated amino acids, asaccharide moiety attached to the amino acid side chain, amino acidscomprising polyethylene glycol or polyether, heavy atom substitutedamino acids, chemically cleavable or photocleavable amino acids, aminoacids with an elongated side chain as compared to natural amino acids(e.g., polyethers or long chain hydrocarbons, e.g., greater than about5, greater than about 10 carbons, etc.), carbon-linked sugar-containingamino acids, amino thioacid containing amino acids, and amino acidscontaining one or more toxic moiety.

In another aspect, the invention provides unnatural amino acids havingthe general structure illustrated by Formula IV below:

An unnatural amino acid having this structure is typically any structurewhere R₁ is a substituent used in one of the twenty natural amino acids(e.g., tyrosine or phenylalanine) and R₂ is a substituent. Thus, thistype of unnatural amino acid can be viewed as a natural amino acidderivative.

In addition to unnatural amino acids that contain the sulfotyrosinestructure shown in FIG. 1, unnatural amino acids can also optionallycomprise modified backbone structures, e.g., as illustrated by thestructures of Formula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids α-aminothiocarboxylates, e.g.,with side chains corresponding to the common twenty natural amino acidsor unnatural side chains. In addition, substitutions at the α-carbonoptionally include L, D, or α-α-disubstituted amino acids such asD-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and thelike. Other structural alternatives include cyclic amino acids, such asproline analogues as well as 3, 4, 6, 7, 8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

In some aspects, the invention utilizes unnatural amino acids in theL-configuration. However, it is not intended that the invention belimited to the use of L-configuration unnatural amino acids. It iscontemplated that the D-enantiomers of these unnatural amino acids alsofind use with the invention.

The unnatural amino acids finding use with the invention is not strictlylimited to the sulfotyrosine unnatural amino acid shown in FIG. 1. Oneof skill in the art will recognize that a wide variety of unnaturalanalogs of naturally occurring amino acids are easily derived. Forexample, but not limited to, unnatural derived from tyrosine are readilyproduced. Tyrosine analogs include, e.g., para-substituted tyrosines,ortho-substituted tyrosines, and meta substituted tyrosines, wherein thesubstituted tyrosine comprises an alkynyl group, acetyl group, a benzoylgroup, an amino group, a hydrazine, an hydroxyamine, a thiol group, acarboxy group, an isopropyl group, a methyl group, a C₆-C₂₀ straightchain or branched hydrocarbon, a saturated or unsaturated hydrocarbon,an O-methyl group, a polyether group, a nitro group, or the like. Inaddition, multiply substituted aryl rings are also contemplated.Glutamine analogs of the invention include, but are not limited to,α-hydroxy derivatives, γ-substituted derivatives, cyclic derivatives,and amide substituted glutamine derivatives. Example phenylalanineanalogs include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, wherein the substituent comprises an alkynyl group, ahydroxy group, a methoxy group, a methyl group, an allyl group, analdehyde, a nitro, a thiol group, or keto group, or the like. Specificexamples of unnatural amino acids include, but are not limited to,sulfotyrosine, p-ethylthiocarbonyl-L-phenylalanine,p-(3-oxobutanoyl)-L-phenylalanine, 1,5-dansyl-alanine, 7-amino-coumarinamino acid, 7-hydroxy-coumarin amino acid, nitrobenzyl-serine,O-(2-nitrobenzyl)-L-tyrosine, p-carboxymethyl-L-phenylalanine,p-cyano-L-phenylalanine, m-cyano-L-phenylalanine, biphenylalanine,3-amino-L-tyrosine, bipyridyl alanine,p-(2-amino-1-hydroxyethyl)-L-phenylalanine,p-isopropylthiocarbonyl-L-phenylalanine, 3-nitro-L-tyrosine andp-nitro-L-phenylalanine. Also, a p-propargyloxyphenylalanine, a3,4-dihydroxy-L-phenyalanine (DHP), a 3,4,6-trihydroxy-L-phenylalanine,a 3,4,5-trihydroxy-L-phenylalanine, 4-nitro-phenylalanine, ap-acetyl-L-phenylalanine, O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a 3-nitro-tyrosine, a3-thiol-tyrosine, a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, afluorinated phenylalanine, an isopropyl-L-phenylalanine, ap-azido-L-phenylalanine, a p-acyl-L-phenylalanine, ap-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, aphosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, ap-amino-L-phenylalanine, and an isopropyl-L-phenylalanine, and the like.The structures of a variety of unnatural amino acids are disclosed inthe references cited herein. See also, WO 2006/110182, filed Oct. 27,2005, entitled “ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVOINCORPORATION OF UNNATURAL AMINO ACIDS.”

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids provided above are commerciallyavailable, e.g., from Sigma (USA) or Aldrich (Milwaukee, Wis., USA).Those that are not commercially available are optionally synthesized asprovided in various publications or using standard methods known tothose of skill in the art. For organic synthesis techniques, see, e.g.,Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition,Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March(Third Edition, 1985, Wiley and Sons, New York); and Advanced OrganicChemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990,Plenum Press, New York). Additional publications describing thesynthesis of unnatural amino acids include, e.g., WO 2002/085923entitled “In vivo incorporation of Unnatural Amino Acids;” Matsoukas etal., (1995) J. Med. Chem., 38, 4660-4669; King and Kidd (1949) “A NewSynthesis of Glutamine and of γ-Dipeptides of Glutamic Acid fromPhthylated Intermediates,” J. Chem. Soc., 3315-3319; Friedman andChatterrji (1959) Synthesis of Derivatives of Glutamine as ModelSubstrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752; Craiget al., (1988) Absolute Configuration of the Enantiomers of 7-Chloro-4[[4-(diethylamino)-1-methylbutyl]amino]quinoline (Chloroquine). J. Org.Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. & Frappier, F. (1991)Glutamine analogues as Potential Antimalarials, Eur. J. Med. Chem. 26,201-5; Koskinen, A. M. P. & Rapoport, H. (1989) Synthesis of4-Substituted Prolines as Conformationally Constrained Amino AcidAnalogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. & Rapoport, H.(1985) Synthesis of Optically Pure Pipecolates from L-Asparagine.Application to the Total Synthesis of (+)-Apovincamine through AminoAcid Decarbonylation and Iminium Ion Cyclization. J. Org. Chem.1989:1859-1866; Barton et al., (1987) Synthesis of Novel a-Amino-Acidsand Derivatives Using Radical Chemistry: Synthesis of L- andD-a-Amino-Adipic Acids, L-a-aminopimelic Acid and AppropriateUnsaturated Derivatives. Tetrahedron Lett. 43:4297-4308; and, Subasingheet al., (1992) Quisqualic acid analogues: synthesis of beta-heterocyclic2-aminopropanoic acid derivatives and their activity at a novelquisqualate-sensitized site. J. Med. Chem. 35:4602-7. See also,International Publication WO 2004/058946, entitled “PROTEIN ARRAYS,”filed on Dec. 22, 2003.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a cell is one issue that is typicallyconsidered when designing and selecting unnatural amino acids, e.g., forincorporation into a protein. For example, the high charge density ofα-amino acids suggests that these compounds are unlikely to be cellpermeable. Natural amino acids are taken up into the cell via acollection of protein-based transport systems often displaying varyingdegrees of amino acid specificity. A rapid screen can be done whichassesses which unnatural amino acids, if any, are taken up by cells.See, e.g., the toxicity assays in, e.g., International Publication WO2004/058946, entitled “PROTEIN ARRAYS,” filed on Dec. 22, 2003; and Liuand Schultz (1999) Progress toward the evolution of an organism with anexpanded genetic code. PNAS 96:4780-4785. Although uptake is easilyanalyzed with various assays, an alternative to designing unnaturalamino acids that are amenable to cellular uptake pathways is to providebiosynthetic pathways to create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, e.g., in acell, the invention provides such methods. For example, biosyntheticpathways for unnatural amino acids are optionally generated in host cellby adding new enzymes or modifying existing host cell pathways.Additional new enzymes are optionally naturally occurring enzymes orartificially evolved enzymes. For example, the biosynthesis ofp-aminophenylalanine (as presented in an example in WO 2002/085923)relies on the addition of a combination of known enzymes from otherorganisms. The genes for these enzymes can be introduced into a cell bytransforming the cell with a plasmid comprising the genes. The genes,when expressed in the cell, provide an enzymatic pathway to synthesizethe desired compound. Examples of the types of enzymes that areoptionally added are provided in the examples below. Additional enzymessequences are found, e.g., in Genbank. Artificially evolved enzymes arealso optionally added into a cell in the same manner. In this manner,the cellular machinery and resources of a cell are manipulated toproduce unnatural amino acids.

Indeed, any of a variety of methods can be used for producing novelenzymes for use in biosynthetic pathways, or for evolution of existingpathways, for the production of unnatural amino acids, in vitro or invivo. Many available methods of evolving enzymes and other biosyntheticpathway components can be applied to the present invention to produceunnatural amino acids (or, indeed, to evolve synthetases to have newsubstrate specificities or other activities of interest). For example,DNA shuffling is optionally used to develop novel enzymes and/orpathways of such enzymes for the production of unnatural amino acids (orproduction of new synthetases), in vitro or in vivo. See, e.g., Stemmer(1994), Rapid evolution of a protein in vitro by DNA shuffling, Nature370(4):389-391; and, Stemmer, (1994), DNA shuffling by randomfragmentation and reassembly: In vitro recombination for molecularevolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751. A relatedapproach shuffles families of related (e.g., homologous) genes toquickly evolve enzymes with desired characteristics. An example of such“family gene shuffling” methods is found in Crameri et al. (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature, 391(6664): 288-291. New enzymes (whether biosyntheticpathway components or synthetases) can also be generated using a DNArecombination procedure known as “incremental-truncation for thecreation of hybrid enzymes” (“ITCHY”), e.g., as described in Ostermeieret al. (1999) “A combinatorial approach to hybrid enzymes independent ofDNA homology” Nature Biotech 17:1205. This approach can also be used togenerate a library of enzyme or other pathway variants which can serveas substrates for one or more in vitro or in vivo recombination methods.See, also, Ostermeier et al. (1999) “Combinatorial Protein Engineeringby Incremental Truncation,” Proc. Natl. Acad. Sci. USA, 96: 3562-67, andOstermeier et al. (1999), “Incremental Truncation as a Strategy in theEngineering of Novel Biocatalysts,” Biological and Medicinal Chemistry,7: 2139-44. Another approach uses exponential ensemble mutagenesis toproduce libraries of enzyme or other pathway variants that are, e.g.,selected for an ability to catalyze a biosynthetic reaction relevant toproducing an unnatural amino acid (or a new synthetase). In thisapproach, small groups of residues in a sequence of interest arerandomized in parallel to identify, at each altered position, aminoacids which lead to functional proteins. Examples of such procedures,which can be adapted to the present invention to produce new enzymes forthe production of unnatural amino acids (or new synthetases) are foundin Delegrave & Youvan (1993) Biotechnology Research 11:1548-1552. In yetanother approach, random or semi-random mutagenesis using doped ordegenerate oligonucleotides for enzyme and/or pathway componentengineering can be used, e.g., by using the general mutagenesis methodsof e.g., Arkin and Youvan (1992) “Optimizing nucleotide mixtures toencode specific subsets of amino acids for semi-random mutagenesis”Biotechnology 10:297-300; or Reidhaar-Olson et al. (1991) “Randommutagenesis of protein sequences using oligonucleotide cassettes”Methods Enzymol. 208:564-86. Yet another approach, often termed a“non-stochastic” mutagenesis, which uses polynucleotide reassembly andsite-saturation mutagenesis can be used to produce enzymes and/orpathway components, which can then be screened for an ability to performone or more synthetase or biosynthetic pathway function (e.g., for theproduction of unnatural amino acids in vivo). See, e.g., Short“NON-STOCHASTIC GENERATION OF GENETIC VACCINES AND ENZYMES” WO 00/46344.

An alternative to such mutational methods involves recombining entiregenomes of organisms and selecting resulting progeny for particularpathway functions (often referred to as “whole genome shuffling”). Thisapproach can be applied to the present invention, e.g., by genomicrecombination and selection of an organism (e.g., an E. coli or othercell) for an ability to produce an unnatural amino acid (or intermediatethereof). For example, methods taught in the following publications canbe applied to pathway design for the evolution of existing and/or newpathways in cells to produce unnatural amino acids in vivo: Patnaik etal. (2002) “Genome shuffling of lactobacillus for improved acidtolerance” Nature Biotechnology, 20(7): 707-712; and Zhang et al. (2002)“Genome shuffling leads to rapid phenotypic improvement in bacteria”Nature, February 7, 415(6872): 644-646.

Other techniques for organism and metabolic pathway engineering, e.g.,for the production of desired compounds are also available and can alsobe applied to the production of unnatural amino acids. Examples ofpublications teaching useful pathway engineering approaches include:Nakamura and White (2003) “Metabolic engineering for the microbialproduction of 1,3 propanediol” Curr. Opin. Biotechnol. 14(5):454-9;Berry et al. (2002) “Application of Metabolic Engineering to improveboth the production and use of Biotech Indigo” J. IndustrialMicrobiology and Biotechnology 28:127-133; Banta et al. (2002)“Optimizing an artificial metabolic pathway: Engineering the cofactorspecificity of Corynebacterium 2,5-diketo-D-gluconic acid reductase foruse in vitamin C biosynthesis” Biochemistry, 41(20), 6226-36; Selivonovaet al. (2001) “Rapid Evolution of Novel Traits in Microorganisms”Applied and Environmental Microbiology, 67:3645, and many others.

Regardless of the method used, typically, the unnatural amino acidproduced with an engineered biosynthetic pathway of the invention isproduced in a concentration sufficient for efficient proteinbiosynthesis, e.g., a natural cellular amount, but not to such a degreeas to significantly affect the concentration of other cellular aminoacids or to exhaust cellular resources. Typical concentrations producedin vivo in this manner are about 10 mM to about 0.05 mM. Once a cell isengineered to produce enzymes desired for a specific pathway and anunnatural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the unnatural amino acid forboth ribosomal protein synthesis and cell growth.

Orthogonal Components for Incorporating Unnatural Amino Acids

The invention provides compositions and methods for producing orthogonalcomponents for incorporating the unnatural amino acid sulfotyrosine (seeFIG. 1) into a growing polypeptide chain in response to a selectorcodon, e.g., an amber stop codon, a nonsense codon, a four or more basecodon, etc., e.g., in vivo. For example, the invention providesorthogonal-tRNAs (O-tRNAs), orthogonal aminoacyl-tRNA synthetases(O-RSs) and pairs thereof. These pairs can be used to incorporate anunnatural amino acid into growing polypeptide chains.

A composition of the invention includes an orthogonal aminoacyl-tRNAsynthetase (O-RS), where the O-RS preferentially aminoacylates an O-tRNAwith sulfotyrosine. In certain embodiments, the O-RS comprises an aminoacid sequence comprising SEQ ID NO: 4, 6, 8 or 10, and conservativevariations thereof. In certain embodiments of the invention, the O-RSpreferentially aminoacylates the O-tRNA over any endogenous tRNA with anthe particular unnatural amino acid, where the O-RS has a bias for theO-tRNA, and where the ratio of O-tRNA charged with an unnatural aminoacid to the endogenous tRNA charged with the same unnatural amino acidis greater than 1:1, and more preferably where the O-RS charges theO-tRNA exclusively or nearly exclusively.

A composition that includes an O-RS can optionally further include anorthogonal tRNA (O-tRNA), where the O-tRNA recognizes a selector codon.Typically, an O-tRNA of the invention includes at least about, e.g., a45%, a 50%, a 60%, a 75%, an 80%, or a 90% or more suppressionefficiency in the presence of a cognate synthetase in response to aselector codon as compared to the suppression efficiency of an O-tRNAcomprising or encoded by a polynucleotide sequence as set forth in thesequence listings (e.g., SEQ ID NO: 1) and examples herein. In oneembodiment, the suppression efficiency of the O-RS and the O-tRNAtogether is, e.g., 5 fold, 10 fold, 15 fold, 20 fold, 25 fold or moregreater than the suppression efficiency of the O-tRNA in the absence ofan O-RS. In some aspects, the suppression efficiency of the O-RS and theO-tRNA together is at least 45% of the suppression efficiency of anorthogonal tyrosyl-tRNA synthetase pair derived from Methanococcusjannaschii.

A composition that includes an O-tRNA can optionally include a cell(e.g., a eubacterial cell, such as an E. coli cell and the like, or aeukaryotic cell such as a yeast cell), and/or a translation system.

A cell (e.g., a eubacterial cell or a yeast cell) comprising atranslation system is also provided by the invention, where thetranslation system includes an orthogonal-tRNA (O-tRNA); an orthogonalaminoacyl-tRNA synthetase (O-RS); and, a sulfotyrosine unnatural aminoacid. Typically, the O-RS preferentially aminoacylates the O-tRNA overany endogenous tRNA with the unnatural amino acid, where the O-RS has abias for the O-tRNA, and where the ratio of O-tRNA charged with theunnatural amino acid to the endogenous tRNA charged with the unnaturalamino acid is greater than 1:1, and more preferably where the O-RScharges the O-tRNA exclusively or nearly exclusively. The O-tRNArecognizes the first selector codon, and the O-RS preferentiallyaminoacylates the O-tRNA with an unnatural amino acid. In oneembodiment, the O-tRNA comprises or is encoded by a polynucleotidesequence as set forth in SEQ ID NO: 1, or a complementary polynucleotidesequence thereof. In one embodiment, the O-RS comprises an amino acidsequence as set forth in SEQ ID NO: 4, 6, 8 or 10, and conservativevariations thereof.

A cell of the invention can optionally further comprise an additionaldifferent O-tRNA/O-RS pair and a second unnatural amino acid, e.g.,where this O-tRNA recognizes a second selector codon and this O-RSpreferentially aminoacylates the corresponding O-tRNA with the secondunnatural amino acid, where the second amino acid is different from thefirst unnatural amino acid. Optionally, a cell of the invention includesa nucleic acid that comprises a polynucleotide that encodes apolypeptide of interest, where the polynucleotide comprises a selectorcodon that is recognized by the O-tRNA.

In certain embodiments, a cell of the invention is a eubacterial cell(such as E. coli), that includes an orthogonal-tRNA (O-tRNA), anorthogonal aminoacyl-tRNA synthetase (O-RS), an unnatural amino acid,and a nucleic acid that comprises a polynucleotide that encodes apolypeptide of interest, where the polynucleotide comprises the selectorcodon that is recognized by the O-tRNA. In certain embodiments of theinvention, the O-RS preferentially aminoacylates the O-tRNA with theunnatural amino acid with an efficiency that is greater than theefficiency with which the O-RS aminoacylates any endogenous tRNA.

In certain embodiments of the invention, an O-tRNA of the inventioncomprises or is encoded by a polynucleotide sequence as set forth in thesequence listings (e.g., SEQ ID NO: 1) or examples herein, or acomplementary polynucleotide sequence thereof. In certain embodiments ofthe invention, an O-RS comprises an amino acid sequence as set forth inthe sequence listings, or a conservative variation thereof. In oneembodiment, the O-RS or a portion thereof is encoded by a polynucleotidesequence encoding an amino acid as set forth in the sequence listings orexamples herein, or a complementary polynucleotide sequence thereof.

The O-tRNA and/or the O-RS of the invention can be derived from any of avariety of organisms (e.g., eukaryotic and/or non-eukaryotic organisms).

Polynucleotides are also a feature of the invention. A polynucleotide ofthe invention (e.g., SEQ ID NO: 5, 7, 9 or 11) includes an artificial(e.g., man-made, and not naturally occurring) polynucleotide comprisinga nucleotide sequence encoding a polypeptide as set forth in thesequence listings herein, and/or is complementary to or thatpolynucleotide sequence. A polynucleotide of the invention can alsoinclude a nucleic acid that hybridizes to a polynucleotide describedabove, under highly stringent conditions, over substantially the entirelength of the nucleic acid. A polynucleotide of the invention alsoincludes a polynucleotide that is, e.g., at least 75%, at least 80%, atleast 90%, at least 95%, at least 98% or more identical to that of anaturally occurring tRNA or corresponding coding nucleic acid (but apolynucleotide of the invention is other than a naturally occurring tRNAor corresponding coding nucleic acid), where the tRNA recognizes aselector codon, e.g., a four base codon. Artificial polynucleotides thatare, e.g., at least 80%, at least 90%, at least 95%, at least 98% ormore identical to any of the above and/or a polynucleotide comprising aconservative variation of any the above, are also included inpolynucleotides of the invention.

Vectors comprising a polynucleotide of the invention are also a featureof the invention. For example, a vector of the invention can include aplasmid, a cosmid, a phage, a virus, an expression vector, and/or thelike. A cell comprising a vector of the invention is also a feature ofthe invention.

Methods of producing components of an O-tRNA/O-RS pair are also featuresof the invention. Components produced by these methods are also afeature of the invention. For example, methods of producing at least onetRNA that is orthogonal to a cell (O-tRNA) include generating a libraryof mutant tRNAs; mutating an anticodon loop of each member of thelibrary of mutant tRNAs to allow recognition of a selector codon,thereby providing a library of potential O-tRNAs, and subjecting tonegative selection a first population of cells of a first species, wherethe cells comprise a member of the library of potential O-tRNAs. Thenegative selection eliminates cells that comprise a member of thelibrary of potential O-tRNAs that is aminoacylated by an aminoacyl-tRNAsynthetase (RS) that is endogenous to the cell. This provides a pool oftRNAs that are orthogonal to the cell of the first species, therebyproviding at least one O-tRNA. An O-tRNA produced by the methods of theinvention is also provided.

In certain embodiments, the methods further comprise subjecting topositive selection a second population of cells of the first species,where the cells comprise a member of the pool of tRNAs that areorthogonal to the cell of the first species, a cognate aminoacyl-tRNAsynthetase, and a positive selection marker. Using the positiveselection, cells are selected or screened for those cells that comprisea member of the pool of tRNAs that is aminoacylated by the cognateaminoacyl-tRNA synthetase and that shows a desired response in thepresence of the positive selection marker, thereby providing an O-tRNA.In certain embodiments, the second population of cells comprise cellsthat were not eliminated by the negative selection.

Methods for identifying an orthogonal-aminoacyl-tRNA synthetase thatcharges an O-tRNA with an unnatural amino acid are also provided. Forexample, methods include subjecting a population of cells of a firstspecies to a selection, where the cells each comprise: 1) a member of aplurality of aminoacyl-tRNA synthetases (RSs), (e.g., the plurality ofRSs can include mutant RSs, RSs derived from a species other than afirst species or both mutant RSs and RSs derived from a species otherthan a first species); 2) the orthogonal-tRNA (O-tRNA) (e.g., from oneor more species); and 3) a polynucleotide that encodes a positiveselection marker and comprises at least one selector codon.

Cells (e.g., a host cell) are selected or screened for those that showan enhancement in suppression efficiency compared to cells lacking orhaving a reduced amount of the member of the plurality of RSs. Theseselected/screened cells comprise an active RS that aminoacylates theO-tRNA. An orthogonal aminoacyl-tRNA synthetase identified by the methodis also a feature of the invention.

Methods of producing a protein in a cell (e.g., in a eubacterial cellsuch as an E. coli cell or the like, or in a yeast cell) having theunnatural amino acid at a selected position are also a feature of theinvention. For example, a method includes growing, in an appropriatemedium, a cell, where the cell comprises a nucleic acid that comprisesat least one selector codon and encodes a protein, providing theunnatural amino acid, and incorporating the unnatural amino acid intothe specified position in the protein during translation of the nucleicacid with the at least one selector codon, thereby producing theprotein. The cell further comprises: an orthogonal-tRNA (O-tRNA) thatfunctions in the cell and recognizes the selector codon; and, anorthogonal aminoacyl-tRNA synthetase (O-RS) that preferentiallyaminoacylates the O-tRNA with the unnatural amino acid. A proteinproduced by this method is also a feature of the invention. Ofparticular interest are methods for producing the sulfated form ofhirudin, which finds use as an anticoagulant.

The invention also provides compositions that include proteins, wherethe proteins comprise sulfotyrosine. In certain embodiments, the proteincomprises an amino acid sequence that is at least 75% identical to thatof a known protein, e.g., hirudin, a therapeutic protein, a diagnosticprotein, an industrial enzyme, or portion thereof. Optionally, thecomposition comprises a pharmaceutically acceptable carrier.

Nucleic Acid and Polypeptide Sequences and Variants

As described herein, the invention provides for polynucleotide sequencesencoding, e.g., O-tRNAs and O-RSs, and polypeptide amino acid sequences,e.g., O-RSs, and, e.g., compositions, systems and methods comprisingsaid polynucleotide or polypeptide sequences. Examples of saidsequences, e.g., O-tRNA and O-RS amino acid and nucleotide sequences aredisclosed herein (see FIG. 7, e.g., SEQ ID NOs: 1 and 4-11). However,one of skill in the art will appreciate that the invention is notlimited to those sequences disclosed herein, e.g., in the Examples andsequence listing. One of skill will appreciate that the invention alsoprovides many related sequences with the functions described herein,e.g., polynucleotides and polypeptides encoding conservative variants ofan O-RS disclosed herein.

The construction and analysis of orthogonal synthetase species (O-RS)that are able to aminoacylate an O-tRNA with an sulfotyrosine aredescribed in Example 1. This Example describes the construction andanalysis of the O-RS species that are able to incorporate the unnaturalamino acid sulfotyrosine.

The invention provides polypeptides (O-RSs) and polynucleotides, e.g.,O-tRNA, polynucleotides that encode O-RSs or portions thereof,oligonucleotides used to isolate aminoacyl-tRNA synthetase clones, etc.Polynucleotides of the invention include those that encode proteins orpolypeptides of interest of the invention with one or more selectorcodon. In addition, polynucleotides of the invention include, e.g., apolynucleotide comprising a nucleotide sequence as set forth in SEQ IDNO: 5, 7, 9 or 11, and a polynucleotide that is complementary to or thatencodes a polynucleotide sequence thereof. A polynucleotide of theinvention also includes any polynucleotide that encodes an O-RS aminoacid sequence comprising SEQ ID NO: 4, 6, 8 or 10. Similarly, anartificial nucleic acid that hybridizes to a polynucleotide indicatedabove under highly stringent conditions over substantially the entirelength of the nucleic acid (and is other than a naturally occurringpolynucleotide) is a polynucleotide of the invention. In one embodiment,a composition includes a polypeptide of the invention and an excipient(e.g., buffer, water, pharmaceutically acceptable excipient, etc.). Theinvention also provides an antibody or antisera specificallyimmunoreactive with a polypeptide of the invention. An artificialpolynucleotide is a polynucleotide that is man made and is not naturallyoccurring.

A polynucleotide of the invention also includes an artificialpolynucleotide that is, e.g., at least 75%, at least 80%, at least 90%,at least 95%, at least 98% or more identical to that of a naturallyoccurring tRNA, (but is other than a naturally occurring tRNA). Apolynucleotide also includes an artificial polynucleotide that is, e.g.,at least 75%, at least 80%, at least 90%, at least 95%, at least 98% ormore identical (but not 100% identical) to that of a naturally occurringtRNA.

In certain embodiments, a vector (e.g., a plasmid, a cosmid, a phage, avirus, etc.) comprises a polynucleotide of the invention. In oneembodiment, the vector is an expression vector. In another embodiment,the expression vector includes a promoter operably linked to one or moreof the polynucleotides of the invention. In another embodiment, a cellcomprises a vector that includes a polynucleotide of the invention.

One of skill will also appreciate that many variants of the disclosedsequences are included in the invention. For example, conservativevariations of the disclosed sequences that yield a functionallyidentical sequence are included in the invention. Variants of thenucleic acid polynucleotide sequences, wherein the variants hybridize toat least one disclosed sequence, are considered to be included in theinvention. Unique subsequences of the sequences disclosed herein, asdetermined by, e.g., standard sequence comparison techniques, are alsoincluded in the invention.

Conservative Variations

Owing to the degeneracy of the genetic code, “silent substitutions”(i.e., substitutions in a nucleic acid sequence which do not result inan alteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence that encodes an amino acid sequence. Similarly,“conservative amino acid substitutions,” where one or a limited numberof amino acids in an amino acid sequence are substituted with differentamino acids with highly similar properties, are also readily identifiedas being highly similar to a disclosed construct. Such conservativevariations of each disclosed sequence are a feature of the presentinvention.

“Conservative variations” of a particular nucleic acid sequence refersto those nucleic acids which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences. One of skillwill recognize that individual substitutions, deletions or additionswhich alter, add or delete a single amino acid or a small percentage ofamino acids (typically less than 5%, more typically less than 4%, 2% or1%) in an encoded sequence are “conservatively modified variations”where the alterations result in the deletion of an amino acid, additionof an amino acid, or substitution of an amino acid with a chemicallysimilar amino acid. Thus, “conservative variations” of a listedpolypeptide sequence of the present invention include substitutions of asmall percentage, typically less than 5%, more typically less than 2% or1%, of the amino acids of the polypeptide sequence, with an amino acidof the same conservative substitution group. Finally, the addition ofsequences which do not alter the encoded activity of a nucleic acidmolecule, such as the addition of a non-functional sequence, is aconservative variation of the basic nucleic acid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art, where one amino acid residue issubstituted for another amino acid residue having similar chemicalproperties (e.g., aromatic side chains or positively charged sidechains), and therefore does not substantially change the functionalproperties of the polypeptide molecule. The following sets forth examplegroups that contain natural amino acids of like chemical properties,where substitutions within a group is a “conservative substitution”.

Conservative Amino Acid Substitutions Nonpolar and/or Polar, PositivelyNegatively Aliphatic Side Uncharged Aromatic Charged Charged Chains SideChains Side Chains Side Chains Side Chains Glycine Serine PhenylalanineLysine Aspartate Alanine Threonine Tyrosine Arginine Glutamate ValineCysteine Tryptophan Histidine Leucine Methionine Isoleucine AsparagineProline Glutamine

Nucleic Acid Hybridization

Comparative hybridization can be used to identify nucleic acids of theinvention, including conservative variations of nucleic acids of theinvention, and this comparative hybridization method is a preferredmethod of distinguishing nucleic acids of the invention. In addition,target nucleic acids which hybridize to a nucleic acid represented bySEQ ID NO: 5, 7, 9 or 11, under high, ultra-high and ultra-ultra highstringency conditions are a feature of the invention. Examples of suchnucleic acids include those with one or a few silent or conservativenucleic acid substitutions as compared to a given nucleic acid sequence.

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least 50% as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at least half as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 5×-10× as high as that observed for hybridization toany of the unmatched target nucleic acids.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, N.Y.), as well asin Current Protocols in Molecular Biology, Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (supplemented through 2006); Hames andHiggins (1995) Gene Probes 1 IRL Press at Oxford University Press,Oxford, England; and Hames and Higgins (1995) Gene Probes 2 IRL Press atOxford University Press, Oxford, England, provide details on thesynthesis, labeling, detection and quantification of DNA and RNA,including oligonucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (for a description of SSC buffer, see, Sambrook etal., Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 2001). Often thehigh stringency wash is preceded by a low stringency wash to removebackground probe signal. An example low stringency wash is 2×SSC at 40°C. for 15 minutes. In general, a signal to noise ratio of 5×(or higher)than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and northern hybridizationsare sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2, “Overview of principles of hybridization and the strategy ofnucleic acid probe assays,” (Elsevier, N.Y.); Hames and Higgins (1995)Gene Probes 1 IRL Press at Oxford University Press, Oxford, England; andHames and Higgins (1995) Gene Probes 2 IRL Press at Oxford UniversityPress, Oxford, England. Stringent hybridization and wash conditions caneasily be determined empirically for any test nucleic acid. For example,in determining stringent hybridization and wash conditions, thehybridization and wash conditions are gradually increased (e.g., byincreasing temperature, decreasing salt concentration, increasingdetergent concentration and/or increasing the concentration of organicsolvents such as formalin in the hybridization or wash), until aselected set of criteria are met. For example, in highly stringenthybridization and wash conditions, the hybridization and wash conditionsare gradually increased until a probe binds to a perfectly matchedcomplementary target with a signal to noise ratio that is at least 5× ashigh as that observed for hybridization of the probe to an unmatchedtarget.

“Very stringent” conditions are selected to be equal to the thermalmelting point (T_(m)) for a particular probe. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. For the purposesof the present invention, generally, “highly stringent” hybridizationand wash conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH.

“Ultra high-stringency” hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched targetnucleic acids. A target nucleic acid which hybridizes to a probe undersuch conditions, with a signal to noise ratio of at least ½ that of theperfectly matched complementary target nucleic acid is said to bind tothe probe under ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids. A target nucleic acid which hybridizesto a probe under such conditions, with a signal to noise ratio of atleast ½ that of the perfectly matched complementary target nucleic acidis said to bind to the probe under ultra-ultra-high stringencyconditions.

Nucleic acids which do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Unique Subsequences

In some aspects, the invention provides a nucleic acid that comprises aunique subsequence in a nucleic acid selected from the sequences ofO-tRNAs and O-RSs disclosed herein. The unique subsequence is unique ascompared to a nucleic acid corresponding to any known O-tRNA or O-RSnucleic acid sequence. Alignment can be performed using, e.g., BLAST setto default parameters. Any unique subsequence is useful, e.g., as aprobe to identify the nucleic acids of the invention or related nucleicacids.

Similarly, the invention includes a polypeptide which comprises a uniquesubsequence in a polypeptide selected from the sequences of O-RSsdisclosed herein. Here, the unique subsequence is unique as compared toa polypeptide corresponding to any of known polypeptide sequence.

The invention also provides for target nucleic acids which hybridizesunder stringent conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from thesequences of O-RSs wherein the unique subsequence is unique as comparedto a polypeptide corresponding to any of the control polypeptides (e.g.,parental sequences from which synthetases of the invention were derived,e.g., by mutation). Unique sequences are determined as noted above.

Sequence Comparison Identity, and Homology

The terms “identical” or “percent identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described below (or otheralgorithms available to persons of skill) or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding an O-tRNA or O-RS, or theamino acid sequence of an O-RS) refers to two or more sequences orsubsequences that have at least about 60%, about 80%, about 90-95%,about 98%, about 99% or more nucleotide or amino acid residue identity,when compared and aligned for maximum correspondence, as measured usinga sequence comparison algorithm or by visual inspection. Such“substantially identical” sequences are typically considered to be“homologous,” without reference to actual ancestry. Preferably, the“substantial identity” exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably, the sequences aresubstantially identical over at least about 150 residues, or over thefull length of the two sequences to be compared.

Proteins and/or protein sequences are “homologous” when they arederived, naturally or artificially, from a common ancestral protein orprotein sequence. Similarly, nucleic acids and/or nucleic acid sequencesare homologous when they are derived, naturally or artificially, from acommon ancestral nucleic acid or nucleic acid sequence. For example, anynaturally occurring nucleic acid can be modified by any availablemutagenesis method to include one or more selector codon. Whenexpressed, this mutagenized nucleic acid encodes a polypeptidecomprising one or more unnatural amino acid. The mutation process can,of course, additionally alter one or more standard codon, therebychanging one or more standard amino acid in the resulting mutant proteinas well. Homology is generally inferred from sequence similarity betweentwo or more nucleic acids or proteins (or sequences thereof). Theprecise percentage of similarity between sequences that is useful inestablishing homology varies with the nucleic acid and protein at issue,but as little as 25% sequence similarity is routinely used to establishhomology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establishhomology. Methods for determining sequence similarity percentages (e.g.,BLASTP and BLASTN using default parameters) are described herein and aregenerally available.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math2:482 (1981), by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol., 48:443 (1970), by the search for similaritymethod of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988),by computerized implementations of these algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.), or by visual inspection(see generally Current Protocols in Molecular Biology, Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., supplemented through2006).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol., 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., J. Mol. Biol.,215:403-410 (1990)). These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc.Natl. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci.USA 90:5873-5787 (1993)). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Mutagenesis and Other Molecular Biology Techniques

Polynucleotide and polypeptides of the invention and used in theinvention can be manipulated using molecular biological techniques.General texts which describe molecular biological techniques includeBerger and Kimmel, “Guide to Molecular Cloning Techniques,” Methods inEnzymology, volume 152 Academic Press, Inc., San Diego, Calif.; Sambrooket al., Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 2001, and CurrentProtocols in Molecular Biology, Ausubel et al., eds., Current Protocols,a joint venture between Greene Publishing Associates, Inc. and JohnWiley & Sons, Inc., (supplemented through 2006). These texts describemutagenesis, the use of vectors, promoters and many other relevanttopics related to, e.g., the generation of genes that include selectorcodons for production of proteins that include unnatural amino acids,orthogonal tRNAs, orthogonal synthetases, and pairs thereof.

Various types of mutagenesis are used in the invention, e.g., to mutatetRNA molecules, to produce libraries of tRNAs, to produce libraries ofsynthetases, to insert selector codons that encode an unnatural aminoacids in a protein or polypeptide of interest. They include but are notlimited to site-directed, random point mutagenesis, homologousrecombination, DNA shuffling or other recursive mutagenesis methods,chimeric construction, mutagenesis using uracil containing templates,oligonucleotide-directed mutagenesis, phosphorothioate-modified DNAmutagenesis, mutagenesis using gapped duplex DNA or the like, or anycombination thereof. Additional suitable methods include point mismatchrepair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like. Mutagenesis, e.g., involving chimeric constructs,is also included in the present invention. In one embodiment,mutagenesis can be guided by known information of the naturallyoccurring molecule or altered or mutated naturally occurring molecule,e.g., sequence, sequence comparisons, physical properties, crystalstructure or the like.

Host cells are genetically engineered (e.g., transformed, transduced ortransfected) with the polynucleotides of the invention or constructswhich include a polynucleotide of the invention, e.g., a vector of theinvention, which can be, for example, a cloning vector or an expressionvector. For example, the coding regions for the orthogonal tRNA, theorthogonal tRNA synthetase, and the protein to be derivatized areoperably linked to gene expression control elements that are functionalin the desired host cell. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both (e.g., shuttle vectors) andselection markers for both prokaryotic and eukaryotic systems. Vectorsare suitable for replication and/or integration in prokaryotes,eukaryotes, or preferably both. See Giliman and Smith, Gene 8:81 (1979);Roberts, et al., Nature. 328:731 (1987); Schneider et al., Protein Expr.Purif., 6435:10 (1995); Berger and Kimmel, “Guide to Molecular CloningTechniques,” Methods in Enzymology, volume 152 Academic Press, Inc., SanDiego, Calif.; Sambrook et al., Molecular Cloning—A Laboratory Manual(3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 2001, and Current Protocols in Molecular Biology, Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (supplemented through2006). The vector can be, for example, in the form of a plasmid, abacterium, a virus, a naked polynucleotide, or a conjugatedpolynucleotide. The vectors are introduced into cells and/ormicroorganisms by standard methods including electroporation (From etal., Proc. Natl. Acad. Sci. USA 82, 5824 (1985), infection by viralvectors, high velocity ballistic penetration by small particles with thenucleic acid either within the matrix of small beads or particles, or onthe surface (Klein et al., Nature 327, 70-73 (1987)), and/or the like.

A highly efficient and versatile single plasmid system was developed forsite-specific incorporation of unnatural amino acids into proteins inresponse to the amber stop codon (UAG) in E. coli. In the new system,the pair of M. jannaschii suppressor tRNAtyr(CUA) and tyrosyl-tRNAsynthetase are encoded in a single plasmid, which is compatible withmost E. coli expression vectors. Monocistronic tRNA operon under controlof proK promoter and terminator was constructed for optimal secondarystructure and tRNA processing. Introduction of a mutated form of glnSpromoter for the synthetase resulted in a significant increase in bothsuppression efficiency and fidelity. Increases in suppression efficiencywere also obtained by multiple copies of tRNA gene as well as by aspecific mutation (D286R) on the synthetase (Kobayashi et al.,“Structural basis for orthogonal tRNA specificities of tyrosyl-tRNAsynthetases for genetic code expansion,” Nat. Struct. Biol.,10(6):425-432 [2003]). The generality of the optimized system was alsodemonstrated by highly efficient and accurate incorporation of severaldifferent unnatural amino acids, whose unique utilities in studyingprotein function and structure were previously proven.

A catalogue of Bacteria and Bacteriophages useful for cloning isprovided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria andBacteriophage (1996) Gherna et al. (eds) published by the ATCC.Additional basic procedures for sequencing, cloning and other aspects ofmolecular biology and underlying theoretical considerations are alsofound in e.g., Sambrook et al., Molecular Cloning—A Laboratory Manual(3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 2001; Current Protocols in Molecular Biology, Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (supplemented through2006), and in Watson et al. (1992) Recombinant DNA, Second Ed.,Scientific American Books, NY. In addition, essentially any nucleic acid(and virtually any labeled nucleic acid, whether standard ornon-standard) can be custom or standard ordered from any of a variety ofcommercial sources, such as the Midland Certified Reagent Company, TheGreat American Gene Company (Ramona, Calif.), ExpressGen Inc. (Chicago,Ill.), Operon Technologies Inc. (Alameda, Calif.) and many others.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, e.g., for cell isolation and culture (e.g., for subsequentnucleic acid isolation) include Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein; Payne et al. (1992) Plant Cell and TissueCulture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg N.Y.) and Atlas and Parks (eds) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla.

Proteins and Polypeptides of Interest

Methods of producing a protein in a cell with an unnatural amino acid ata specified position are also a feature of the invention. For example, amethod includes growing, in an appropriate medium, the cell, where thecell comprises a nucleic acid that comprises at least one selector codonand encodes a protein; and, providing the unnatural amino acid; wherethe cell further comprises: an orthogonal-tRNA (O-tRNA) that functionsin the cell and recognizes the selector codon; and, an orthogonalaminoacyl-tRNA synthetase (O-RS) that preferentially aminoacylates theO-tRNA with the unnatural amino acid. A protein produced by this methodis also a feature of the invention.

In certain embodiments, the O-RS comprises a bias for the aminoacylationof the cognate O-tRNA over any endogenous tRNA in an expression system.The relative ratio between O-tRNA and endogenous tRNA that is charged bythe O-RS, when the O-tRNA and O-RS are present at equal molarconcentrations, is greater than 1:1, preferably at least about 2:1, morepreferably 5:1, still more preferably 10:1, yet more preferably 20:1,still more preferably 50:1, yet more preferably 75:1, still morepreferably 95:1, 98:1, 99:1, 100:1, 500:1, 1,000:1, 5,000:1 or higher.

The invention also provides compositions that include proteins, wherethe proteins comprise an unnatural amino acid. In certain embodiments,the protein comprises an amino acid sequence that is at least 75%identical to that of a therapeutic protein, a diagnostic protein, anindustrial enzyme, or portion thereof.

The compositions of the invention and compositions made by the methodsof the invention optionally are in a cell. The O-tRNA/O-RS pairs orindividual components of the invention can then be used in a hostsystem's translation machinery, which results in an unnatural amino acidbeing incorporated into a protein. International Publication Numbers WO2004/094593, filed Apr. 16, 2004, entitled “EXPANDING THE EUKARYOTICGENETIC CODE,” and WO 2002/085923, entitled “IN VIVO INCORPORATION OFUNNATURAL AMINO ACIDS,” describe this process, and are incorporatedherein by reference. For example, when an O-tRNA/O-RS pair is introducedinto a host, e.g., an Escherichia coli cell, the pair leads to the invivo incorporation of an unnatural amino acid such as sulfotyrosine intoa protein in response to a selector codon. The unnatural amino acid thatis added to the system can be a synthetic amino acid, such as aderivative of a phenylalanine or tyrosine, which can be exogenouslyadded to the growth medium. Optionally, the compositions of the presentinvention can be in an in vitro translation system, or in an in vivosystem(s).

A cell of the invention provides the ability to synthesize proteins thatcomprise unnatural amino acids in large useful quantities. In someaspects, the composition optionally includes, e.g., at least 10micrograms, at least 50 micrograms, at least 75 micrograms, at least 100micrograms, at least 200 micrograms, at least 250 micrograms, at least500 micrograms, at least 1 milligram, at least 10 milligrams or more ofthe protein that comprises an unnatural amino acid, or an amount thatcan be achieved with in vivo protein production methods (details onrecombinant protein production and purification are provided herein). Inanother aspect, the protein is optionally present in the composition ata concentration of, e.g., at least 10 micrograms of protein per liter,at least 50 micrograms of protein per liter, at least 75 micrograms ofprotein per liter, at least 100 micrograms of protein per liter, atleast 200 micrograms of protein per liter, at least 250 micrograms ofprotein per liter, at least 500 micrograms of protein per liter, atleast 1 milligram of protein per liter, or at least 10 milligrams ofprotein per liter or more, in, e.g., a cell lysate, a buffer, apharmaceutical buffer, or other liquid suspension (e.g., in a volume of,e.g., anywhere from about 1 mL to about 100 L). The production of largequantities (e.g., greater that that typically possible with othermethods, e.g., in vitro translation) of a protein in a cell including atleast one unnatural amino acid is a feature of the invention.

The incorporation of an unnatural amino acid can be done to, e.g.,tailor changes in protein structure and/or function, e.g., to changesize, acidity, nucleophilicity, hydrogen bonding, hydrophobicity,accessibility of protease target sites, target to a moiety (e.g., for aprotein array), incorporation of labels or reactive groups, etc.Proteins that include an unnatural amino acid can have enhanced or evenentirely new catalytic or physical properties. For example, thefollowing properties are optionally modified by inclusion of anunnatural amino acid into a protein: toxicity, biodistribution,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic ability, half-life (e.g., serumhalf-life), ability to react with other molecules, e.g., covalently ornoncovalently, and the like. The compositions including proteins thatinclude at least one unnatural amino acid are useful for, e.g., noveltherapeutics, diagnostics, catalytic enzymes, industrial enzymes,binding proteins (e.g., antibodies), and e.g., the study of proteinstructure and function. See, e.g., Dougherty, (2000) Unnatural AminoAcids as Probes of Protein Structure and Function, Current Opinion inChemical Biology, 4:645-652.

In some aspects of the invention, a composition includes at least oneprotein with at least one, e.g., at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, or at least ten or more unnatural amino acids. The unnaturalamino acids can be the same or different, e.g., there can be 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 or more different sites in the protein thatcomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnaturalamino acids. In another aspect, a composition includes a protein with atleast one, but fewer than all, of a particular amino acid present in theprotein is an unnatural amino acid. For a given protein with more thanone unnatural amino acids, the unnatural amino acids can be identical ordifferent (e.g., the protein can include two or more different types ofunnatural amino acids, or can include two of the same unnatural aminoacid). For a given protein with more than two unnatural amino acids, theunnatural amino acids can be the same, different or a combination of amultiple unnatural amino acid of the same kind with at least onedifferent unnatural amino acid.

Essentially any protein (or portion thereof) that includes an unnaturalamino acid (and any corresponding coding nucleic acid, e.g., whichincludes one or more selector codons) can be produced using thecompositions and methods herein. No attempt is made to identify thehundreds of thousands of known proteins, any of which can be modified toinclude one or more unnatural amino acid, e.g., by tailoring anyavailable mutation methods to include one or more appropriate selectorcodon in a relevant translation system. Common sequence repositories forknown proteins include GenBank EMBL, DDBJ and the NCBI. Otherrepositories can easily be identified by searching the internet.

Typically, the proteins are, e.g., at least 60%, at least 70%, at least75%, at least 80%, at least 90%, at least 95%, or at least 99% or moreidentical to any available protein (e.g., a therapeutic protein, adiagnostic protein, an industrial enzyme, or portion thereof, and thelike), and they comprise one or more unnatural amino acid. Examples oftherapeutic, diagnostic, and other proteins that can be modified tocomprise one or more unnatural amino acid can be found, but not limitedto, those in International Publications WO 2004/094593, filed Apr. 16,2004, entitled “EXPANDING THE EUKARYOTIC GENETIC CODE;” and, WO2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS.”Examples of therapeutic, diagnostic, and other proteins that can bemodified to comprise one or more unnatural amino acids include, but arenot limited to, e.g., hirudin, Alpha-1 antitrypsin, Angiostatin,Antihemolytic factor, antibodies (further details on antibodies arefound below), Apolipoprotein, Apoprotein, Atrial natriuretic factor,Atrial natriuretic polypeptide, Atrial peptides, C-X-C chemokines (e.g.,T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1,PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractantprotein-1, Monocyte chemoattractant protein-2, Monocyte chemoattractantprotein-3, Monocyte inflammatory protein-1 alpha, Monocyte inflammatoryprotein-1beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065,T64262), CD40 ligand, C-kit Ligand, Collagen, Colony stimulating factor(CSF), Complement factor 5a, Complement inhibitor, Complement receptor1, cytokines, (e.g., epithelial Neutrophil Activating Peptide-78,GROα/MGSA, GROβ, GROγ, MIP-1α, MIP-16, MCP-1), Epidermal Growth Factor(EGF), Erythropoietin (“EPO”), Exfoliating toxins A and B, Factor IX,Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor (FGF),Fibrinogen, Fibronectin, G-CSF, GM-CSF, Glucocerebrosidase,Gonadotropin, growth factors, Hedgehog proteins (e.g., Sonic, Indian,Desert), Hemoglobin, Hepatocyte Growth Factor (HGF), Hirudin, Humanserum albumin, Insulin, Insulin-like Growth Factor (IGF), interferons(e.g., IFN-α, IFN-β, IFN-γ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, etc.), KeratinocyteGrowth Factor (KGF), Lactoferrin, leukemia inhibitory factor,Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), oncostatin M,Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones(e.g., Human Growth Hormone), Pleiotropin, Protein A, Protein G,Pyrogenic exotoxins A, B, and C, Relaxin, Renin, SCF, Soluble complementreceptor I, Soluble I-CAM 1, Soluble interleukin receptors (IL-1, 2, 3,4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15), Soluble TNF receptor,Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigens,i.e., Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED,SEE), Superoxide dismutase (SOD), Toxic shock syndrome toxin (TSST-1),Thymosin alpha 1, Tissue plasminogen activator, Tumor necrosis factorbeta (TNF beta), Tumor necrosis factor receptor (TNFR), Tumor necrosisfactor-alpha (TNF alpha), Vascular Endothelial Growth Factor (VEGEF),Urokinase and many others.

One class of proteins that can be made using the compositions andmethods for in vivo incorporation of unnatural amino acids describedherein includes transcriptional modulators or a portion thereof. Exampletranscriptional modulators include genes and transcriptional modulatorproteins that modulate cell growth, differentiation, regulation, or thelike. Transcriptional modulators are found in prokaryotes, viruses, andeukaryotes, including fungi, plants, yeasts, insects, and animals,including mammals, providing a wide range of therapeutic targets. Itwill be appreciated that expression and transcriptional activatorsregulate transcription by many mechanisms, e.g., by binding toreceptors, stimulating a signal transduction cascade, regulatingexpression of transcription factors, binding to promoters and enhancers,binding to proteins that bind to promoters and enhancers, unwinding DNA,splicing pre-mRNA, polyadenylating RNA, and degrading RNA.

One class of proteins of the invention (e.g., proteins with one or moreunnatural amino acids) include biologically active proteins such ashirudin, cytokines, inflammatory molecules, growth factors, theirreceptors, and oncogene products, e.g., interleukins (e.g., IL-1, IL-2,IL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α,TGF-β, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, andhyalurin/CD44; signal transduction molecules and corresponding oncogeneproducts, e.g., Mos, Ras, Raf, and Met; and transcriptional activatorsand suppressors, e.g., p53, Tat, Fos, Myc, Jun, Myb, Rel, and steroidhormone receptors such as those for estrogen, progesterone,testosterone, aldosterone, the LDL receptor ligand and corticosterone.

Enzymes (e.g., industrial enzymes) or portions thereof with at least oneunnatural amino acid are also provided by the invention. Examples ofenzymes include, but are not limited to, e.g., amidases, amino acidracemases, acylases, dehalogenases, dioxygenases, diarylpropaneperoxidases, epimerases, epoxide hydrolases, esterases, isomerases,kinases, glucose isomerases, glycosidases, glycosyl transferases,haloperoxidases, monooxygenases (e.g., p450s), lipases, ligninperoxidases, nitrile hydratases, nitrilases, proteases, phosphatases,subtilisins, transaminase, and nucleases.

Many of these proteins are commercially available (See, e.g., the SigmaBioSciences 2002 catalogue and price list), and the correspondingprotein sequences and genes and, typically, many variants thereof, arewell-known (see, e.g., Genbank). Any of them can be modified by theinsertion of one or more unnatural amino acid according to theinvention, e.g., to alter the protein with respect to one or moretherapeutic, diagnostic or enzymatic properties of interest. Examples oftherapeutically relevant properties include serum half-life, shelfhalf-life, stability, immunogenicity, therapeutic activity,detectability (e.g., by the inclusion of reporter groups (e.g., labelsor label binding sites) in the unnatural amino acids), reduction of LD₅₀or other side effects, ability to enter the body through the gastrictract (e.g., oral availability), or the like. Examples of diagnosticproperties include shelf half-life, stability, diagnostic activity,detectability, or the like. Examples of relevant enzymatic propertiesinclude shelf half-life, stability, enzymatic activity, productioncapability, or the like.

A variety of other proteins can also be modified to include one or moreunnatural amino acid using compositions and methods of the invention.For example, the invention can include substituting one or more naturalamino acids in one or more vaccine proteins with an unnatural aminoacid, e.g., in proteins from infectious fungi, e.g., Aspergillus,Candida species; bacteria, particularly E. coli, which serves a modelfor pathogenic bacteria, as well as medically important bacteria such asStaphylococci (e.g., aureus), or Streptococci (e.g., pneumoniae);protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba)and flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.);viruses such as (+) RNA viruses (examples include Poxviruses e.g.,vaccinia; Picornaviruses, e.g. polio; Togaviruses, e.g., rubella;Flaviviruses, e.g., HCV; and Coronaviruses), (−) RNA viruses (e.g.,Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses,e.g., influenza; Bunyaviruses; and Arenaviruses), dsDNA viruses(Reoviruses, for example), RNA to DNA viruses, i.e., Retroviruses, e.g.,HIV and HTLV, and certain DNA to RNA viruses such as Hepatitis B.

Agriculturally related proteins such as insect resistance proteins(e.g., the Cry proteins), starch and lipid production enzymes, plant andinsect toxins, toxin-resistance proteins, Mycotoxin detoxificationproteins, plant growth enzymes (e.g., Ribulose 1,5-BisphosphateCarboxylase/Oxygenase, “RUBISCO”), lipoxygenase (LOX), andPhosphoenolpyruvate (PEP) carboxylase are also suitable targets forunnatural amino acid modification.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods well-known to one of skill in the art and described hereinunder “Mutagenesis and Other Molecular Biology Techniques” to include,e.g., one or more selector codon for the incorporation of an unnaturalamino acid. For example, a nucleic acid for a protein of interest ismutagenized to include one or more selector codon, providing for theinsertion of the one or more unnatural amino acids. The inventionincludes any such variant, e.g., mutant, versions of any protein, e.g.,including at least one unnatural amino acid. Similarly, the inventionalso includes corresponding nucleic acids, i.e., any nucleic acid withone or more selector codon that encodes one or more unnatural aminoacid.

To make a protein that includes an unnatural amino acid, one can usehost cells and organisms that are adapted for the in vivo incorporationof the unnatural amino acid via orthogonal tRNA/RS pairs. Host cells aregenetically engineered (e.g., transformed, transduced or transfected)with one or more vectors that express the orthogonal tRNA, theorthogonal tRNA synthetase, and a vector that encodes the protein to bederivatized. Each of these components can be on the same vector, or eachcan be on a separate vector, or two components can be on one vector andthe third component on a second vector. The vector can be, for example,in the form of a plasmid, a bacterium, a virus, a naked polynucleotide,or a conjugated polynucleotide.

Defining Polypeptides by Immunoreactivity

Because the polypeptides of the invention provide a variety of newpolypeptide sequences (e.g., polypeptides comprising unnatural aminoacids in the case of proteins synthesized in the translation systemsherein, or, e.g., in the case of the novel synthetases, novel sequencesof standard amino acids), the polypeptides also provide new structuralfeatures which can be recognized, e.g., in immunological assays. Thegeneration of antisera, which specifically bind the polypeptides of theinvention, as well as the polypeptides which are bound by such antisera,are a feature of the invention. The term “antibody,” as used herein,includes, but is not limited to a polypeptide substantially encoded byan immunoglobulin gene or immunoglobulin genes, or fragments thereofwhich specifically bind and recognize an analyte (antigen). Examplesinclude polyclonal, monoclonal, chimeric, and single chain antibodies,and the like. Fragments of immunoglobulins, including Fab fragments andfragments produced by an expression library, including phage display,are also included in the term “antibody” as used herein. See, e.g.,Paul, Fundamental Immunology, 4th Ed., 1999, Raven Press, New York, forantibody structure and terminology.

In order to produce antisera for use in an immunoassay, one or more ofthe immunogenic polypeptides is produced and purified as describedherein. For example, recombinant protein can be produced in arecombinant cell. An inbred strain of mice (used in this assay becauseresults are more reproducible due to the virtual genetic identity of themice) is immunized with the immunogenic protein(s) in combination with astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see, e.g., Harlow and Lane (1988) Antibodies ALaboratory Manual, Cold Spring Harbor Publications, New York, for astandard description of antibody generation, immunoassay formats andconditions that can be used to determine specific immunoreactivity.Additional details on proteins, antibodies, antisera, etc. can be foundin International Publication Numbers WO 2004/094593, entitled “EXPANDINGTHE EUKARYOTIC GENETIC CODE;” WO 2002/085923, entitled “IN VIVOINCORPORATION OF UNNATURAL AMINO ACIDS;” WO 2004/035605, entitled“GLYCOPROTEIN SYNTHESIS;” and WO 2004/058946, entitled “PROTEIN ARRAYS.”

Use of O-tRNA and O-RS and O-tRNA/O-RS Pairs

The compositions of the invention and compositions made by the methodsof the invention optionally are in a cell. The O-tRNA/O-RS pairs orindividual components of the invention can then be used in a hostsystem's translation machinery, which results in an unnatural amino acidbeing incorporated into a protein. International Publication Number WO2002/085923 by Schultz, et al., entitled “IN VIVO INCORPORATION OFUNNATURAL AMINO ACIDS,” describes this process and is incorporatedherein by reference. For example, when an O-tRNA/O-RS pair is introducedinto a host, e.g., Escherichia coli or yeast, the pair leads to the invivo incorporation of an unnatural amino acid, which can be exogenouslyadded to the growth medium, into a protein, e.g., a myoglobin testprotein or a therapeutic protein, in response to a selector codon, e.g.,an amber nonsense codon. Optionally, the compositions of the inventioncan be in an in vitro translation system, or in a cellular in vivosystem(s). Proteins with the unnatural amino acid can be used in any ofa wide range of applications. For example, the unnatural moietyincorporated into a protein can serve as a target for any of a widerange of modifications, for example, crosslinking with other proteins,with small molecules such as labels or dyes and/or biomolecules. Withthese modifications, incorporation of the unnatural amino acid canresult in improved therapeutic proteins and can be used to alter orimprove the catalytic function of enzymes. In some aspects, theincorporation and subsequent modification of an unnatural amino acid ina protein can facilitate studies on protein structure, interactions withother proteins, and the like.

Kits

Kits are also a feature of the invention. For example, a kit forproducing a protein that comprises at least one unnatural amino acid ina cell is provided, where the kit includes at least one containercontaining a polynucleotide sequence encoding an O-tRNA, and/or anO-tRNA, and/or a polynucleotide sequence encoding an O-RS, and/or anO-RS. In one embodiment, the kit further includes the unnatural aminoacid sulfotyrosine. In another embodiments, the kit further comprisesinstructional materials for producing the protein and/or a host cell.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. One of skill will recognize a variety of non-criticalparameters that may be altered without departing from the scope of theclaimed invention. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and scope of the appended claims.

Example 1 Genetic Selection of Mutant Synthetases Specific forSulfotyrosine

Methodologies that allow the systematic addition of unnatural aminoacids to the genetic codes of E. coli (Wang et al., “Expanding thegenetic code of Escherichia coli,” Science 292:498-500 (2001)), yeast(Chin et al., “An expanded eukaryotic genetic code,” Science 301:964-967(2003)) and mammalian cells (Zhang et al., “Selective incorporation of5-hydroxytryptophan into proteins in mammalian cells,” Proc Natl AcadSci USA 101:8882-8887 (2004)) have been previously reported. Suchmethods are based on the evolution of a nonsense suppressor tRNA/aaRSpair that has the property of orthogonality, defined as the ability toselectively incorporate a given amino acid in response to a unique codonwithout cross-reacting with endogenous host tRNAs, aminoacyl-tRNAsynthetases, or amino acids.

To generate an orthogonal tRNA/aaRS pair that uniquely insertssulfotyrosine (FIG. 1), a library of active site mutants of theMethanococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS), whichspecifically charges an engineered M. jannaschii nonsense suppressor(MjtRNA^(Tyr) _(CUA)) not recognized by E. coli synthetases (Wang etal., “Expanding the genetic code of Escherichia coli,” Science292:498-500 (2001)) was used. This library, whose design and generationare described elsewhere (Bose et al., “The incorporation of aphotoisomerizable amino acid into proteins in E. coli,” J Am Chem Soc128:388-389 (2006)) was subjected to a series of positive and negativeselections (3 positive and 2 negative). Survival in the positiveselection is contingent upon suppression of an amber mutation in thechloramphenicol acetyltransferase (CAT) gene in the presence of 2 mMsulfotyrosine; survival in the negative selection is contingent uponinadequate suppression of three amber mutations in a gene encoding thetoxic barnase protein in the absence of sulfotyrosine (Wang et al.,“Expanding the genetic code of Escherichia coli,” Science 292:498-500(2001)). Clones survive through both positive and negative rounds ofselection only if they uniquely incorporate sulfotyrosine in response tothe amber codon.

Following these selections, numerous clones were identified that allowedcells harboring the CAT gene with an amber mutation at the permissivesite 112 to survive on 130 μg/mL chloramphenicol in the presence of 2 mMsulfotyrosine. In the absence of sulfotyrosine, the same cells did notgrow on 20 μg/mL chloramphenicol, consistent with efficientsulfotyrosine incorporation with little to no background fromincorporation of endogenous amino acids. Sequencing of the candidatemutant synthetase clones (termed STyrRS) revealed four differentsynthetase clones, each of which fulfill the criteria for an orthogonaltranslation system. Clone 1 was predominant (Tyr32Leu, Leu65Pro,Asp158Gly, Ile159Cys, Leu162Lys). The nucleotide and amino acidsequences of each of these clones and the wild-type species is providedin FIG. 7.

Mj tyrosyl-tRNA synthetase amino acid (and corresponding codon) SEQ ID32 65 155 158 159 162 NO: wild-type Tyr Leu Gln Asp Ile Leu 2 (TAC)(TTG) (CAG) (GAT) (ATT) (TTA) (3) Clone 1 Leu Pro Gln Gly Cys Lys 4(CTG) (CCT) (CAG) (GGT) (TGT) (AAG) (5) Clone 2 Leu Pro Gln Gly Thr Lys6 (CTG) (CCG) (CAG) (GGT) (ACT) (AAG) (7) Clone 3 Leu Pro Glu Gly CysLys 8 (CTG) (CCT) (GAG) (GGT) (TGT) (AAG) (9) Clone 4 Leu Pro Gln GlyIle Lys 10  (CTG) (CCG) (CAG) (GGT) (ATT) (AAG) (11) 

It is possible to assign possible roles for these mutations,particularly Lys162, which likely forms a salt-bridge interaction withthe sulfotyrosine SO₃ ⁻. Leu32 and Gly158 may accommodate the larger SO₃⁻ group and remove affinity for endogenous tyrosine (Tyr32 and Asp158are involved in hydrogen bonding to the tyrosine phenolic group inwild-type enzyme). Replacement of anionic Asp158 by Gly possiblyobviates unfavorable electrostatic interactions with sulfotyrosine.However, an understanding of the mechanism or roles of the varioussubstituted positions is not required to make or use the invention.

Detailed Methodology for Selection of Sulfotyrosine Amino-Acyl tRNASynthetase

To select for STyrRS, a MjTyrRS active site library housed in the pBKvector (pBK-lib) was used (Bose et al., “The incorporation of aphotoisomerizable amino acid into proteins in E. coli,” J Am Chem Soc128:388-389 (2006)). DH10B cells harboring pRep, a positive selectionplasmid containing an engineered MjtRNA^(Tyr) _(CUA), a chloramphenicolacetyltransferase gene with an amber codon introduced at position 112 (apermissive site), and a tetracycline resistance marker, were transformedwith pBK-lib and plated on GMML agar plates supplemented with 2 mMsulfotyrosine (Senn Chemicals) and 68 μg/mL chloramphenicol. After 72hours at 37° C., the plates were scraped and the pBK-lib vectors wereextracted.

This collection of library plasmids was then used to transform DH10Bcells harboring pNeg, a negative selection plasmid containing anengineered MjtRNA^(Tyr) _(CUA), a toxic barnase gene with three ambercodons introduced, and a chloramphenicol resistance marker. The cellswere plated on LB agar plates containing no sulfotyrosine and grown at37° C. for 12 hours after which the pBK-lib vectors were extracted fromthe surviving cells. This cycle of positive and negative selection wasrepeated once, and the selected pBK-lib vectors were subsequentlytransformed into DH10B cells harboring pRep and replica plated on GMMLagar plates with and without sulfotyrosine. Those cells that grew onplates containing 130 μg/mL chloramphenicol in the presence ofsulfotyrosine but did not grow on plates containing 20 μg/mLchloramphenicol in the absence of sulfotyrosine were considered stronghits.

These hits were picked and the orthogonality of their correspondingsynthetases was confirmed by expressing Z-domain protein containing anamber codon at position 7 in the presence and absence of sulfotyrosine.Orthogonal synthetases were those that allowed full-length Z-domainexpression only in the presence of sulfotyrosine. MALDI-TOF was used toconfirm that sulfotyrosine was indeed incorporated in the full-lengthZ-domain.

Example 2 Expression and Characterization of a Mutant Model ProteinContaining Sulfotyrosine

To verify unique incorporation of sulfotyrosine by the selectedsynthetase STyrRS, an amber mutant (residue 7) of a C-terminal His₆ tagZ-domain protein was expressed in E. coli harboring plasmids for theamber mutant Z-domain, MjtRNA^(Tyr) _(CUA), and STyrRS (clone 1).Polyacrylamide gel electrophoresis (PAGE) analysis after Ni-NTApurification showed a strong band for Z-domain only when protein wasexpressed in media containing 2 mM sulfotyrosine—no band was observed inthe absence of sulfotyrosine, confirming the dependence of ambersuppression on sulfotyrosine (FIG. 4A).

For further characterization, MALDI-TOF analysis was performed on thepurified mutant Z-domain. It should be noted that MALDI-TOF and ESIanalyses of tyrosine-sulfated proteins result in partial loss ofsulfate, the extent of which depends on the severity of the conditions(22, 23). Therefore, mild positive-ion mode conditions with a moderatepH matrix (2,4,6-trihydroxy-acetophenone) were used, under which apredominant [M+H] peak of 7876 Da (M_(theoretical)=7877.5 Da)corresponding to Z-domain containing a single sulfotyrosine and lackingmethionine appeared. We also observed a small (<10%) [M+H] peak of 7798Da (M_(theoretical)=7797.5 Da) that is the result of loss of sulfateduring MALDI-TOF, leaving tyrosine (FIG. 4B). Although these massspectrometry data alone do not rule out background tyrosineincorporation by STyrRS, we can do so on the basis of the PAGE gelanalysis. STyrRS thus uniquely incorporates sulfotyrosine, allowing therecombinant expression of sulfated proteins in bacteria.

Example 3 Expression of a Sulfated Model Protein (Hirudin) Derived froma Higher Organism

Whether this orthogonal system for the production of sulfated proteinscould be used to generate a selectively sulfated native protein normallybiosynthesized only in higher organisms was examined. For this, we chosethe protein hirudin, which is sulfated at tyrosine position 63. Hirudin,secreted by the medicinal leech Hirudo medicinalis, is the most potentnatural inhibitor of thrombin, and its recombinant form is clinicallyadministered as an anticoagulant. However, recombinant expression ofhirudin in E. coli and yeast used for commercial production of the drugyields the non-sulfated form (desulfo-hirudin) due to the lack ofrequisite sulfotransferases in those organisms (Markwardt, “Hirudin asalternative anticoagulant—a historical review,” Semin Thromb Hemost 28,405-414 (2002)). Although desulfo-hirudin is still an effective thrombininhibitor, its affinity for human thrombin is at least an order ofmagnitude lower than that of sulfo-hirudin, which has a K_(i) around 20fM (Braun et al., “Use of site-directed mutagenesis to investigate thebasis for the specificity of hirudin,” Biochemistry 27, 6517-6522(1988)).

To express sulfo-hirudin, the STyrRS (clone 1) gene was cloned into thepSup vector backbone containing six copies of MjRNA^(Tyr) _(CUA) withoptimized promoters (Ryu and Schultz, “Efficient incorporation ofunnatural amino acids into proteins in Escherichia coli,” Nat Methods3:263-265 (2006)). The hirudin gene with an amber codon at position 63and a gIII periplasmic signal sequence was synthesized and inserted intothe pBAD vector. After cotransformation of DH10B E. coli cells with bothplasmids, shake-flask expression in liquid glycerol minimal media (GMML)supplemented with 10 mM sulfotyrosine was carried out. Since hirudin issmall, direction into the periplasm effectively results in secretion;therefore, the sulfo-hirudin was purified directly from the concentratedmedia by FPLC using a Q Sepharose anion-exchange column followed bysize-exclusion chromatography to give a yield of 5 mg/L. For comparison,desulfo-hirudin with tyrosine encoded at position 63 was similarlyexpressed and purified with a yield of 12 mg/L.

Detailed Methodology for Cloning, Expression and Purification ofSulfo-Hirudin and Desulfo-Hirudin

The gene corresponding to [Leu¹, Thr²]-63-desulfo-hirudin (commerciallyknow as Lepirudin (Refludan®)) fused to a gIII periplasmic signalsequence for secretion was synthesized by BlueHeron® with ExpressionOptimization. This gene was inserted into the pBAD vector (Invitrogen)to yield pBAD-Hirudin under the control of the araBAD promoter.Quickchange (Stratagene) site-directed mutagenesis was used to introduceTAG at position 63 of the Lepirudin gene to yield pBAD-HirudinTAG forexpression of sulfo-hirudin.

The gene corresponding to the selected STyrRS (clone 1) was insertedinto the pSup vector between sites PstI and NdeI under the control ofthe glnS promoter to yield pSup-STyrRS. The pSup-STyrRS also containssix copies of the engineered MjRNA^(Tyr) _(CUA) under control of theproK promoter.

Electro-competent DH10B cells cotransformed with pSup-STyrRS andpBAD-HirudinTAG were grown in GMML medium with 50 μg/ml ampicillin, 20μg/ml of chloramphenicol and 10 mM sulfotyrosine at 37° C. When cellsreached an OD₆₀₀ of 0.6, L-arabinose was added to a final concentrationof 0.2% to induce protein expression. Cells were grown for an additional24 hours at 37° C. The cells were pelleted and the media wasconcentrated using a stirred cell apparatus.

The concentrated media was dialyzed against water and applied to ananion exchange column (HiLoad 26/10 Q Sepharose, GE Healthcare)previously equilibrated with 50 mM Tris-HCl, 1 mM EDTA, and 10 mMβ-mercaptoethanol, pH 7.4. The proteins were eluted with a lineargradient from 0.025 to 1 M NaCl. Peak fractions were analyzed by PAGE.Fractions from a major peak that eluted at 0.3 M NaCl were pooledtogether, concentrated, dialyzed against water, and applied to gelfiltration (Superdex 200 10/300 GL, GE Healthcare). Proteins were elutedwith Tris-buffered saline (25 mM Tris-HCl, 125 mM NaCl, and 2 mM KCl, pH7.6). The final sulfo-hirudin concentration was determined by titrationagainst 1 nM human α-thrombin (Diapharma) using 50 μM of the fluorogenicsubstrate of thrombin Boc-Asp(OBzl)-Pro-Arg-MCA (Peptides International,Inc.) to measure thrombin activity. This assumes a 1:1 stoichiometricinhibition of thrombin by hirudin, which is valid under theconcentrations used as dictated by tight-binding kinetics (Szedlacsekand Duggleby, “Kinetics of slow and tight-binding inhibitors,” MethodsEnzymol 249:144-180 (1995)). Similar procedures were used to express,purify, and quantify [Leu¹, Thr²]-63-desulfo-hirudin.

Example 4 Characterization of a Genetically Encoded Sulfated Hirudin

The resulting hirudins described in the previous example werecharacterized by PAGE analysis and each was present as a single band.Sulfo-hirudin could be distinguished from desulfo-hirudin since theformer migrates farther than the latter to afford a gel shift (FIG. 2).MALDI-TOF analysis showed the correct [M+H] masses for bothsulfo-hirudin (7059 Da; M_(theoretical)=7059.5 Da) and desulfo-hirudin(6979 Da; M_(theoretical)=6979.5 Da) with two peaks in the sulfo-hirudincase as loss of sulfate results in a minor [M+H-80] signal (see FIG. 5).

To further verify that this second peak resulted solely from massspectral analysis, two experiments were conducted. First, the fact thatelution of sulfo-hirudin from the anion-exchange column occurs at a 10%greater ionic strength than elution of desulfo-hirudin under the samegradient conditions was exploited, which would allow complete separationof the two hirudins had they been simultaneously present. (This wasconfirmed by spiking sulfo-hirudin with desulfo-hirudin.) Since nodesulfo-hirudin peak was observed in the sulfo-hirudin anion-exchangepurification as determined by the lack of a desulfo-hirudin peak in themass spectra of the corresponding eluted fractions, we conclude that nodesulfo-hirudin was produced when sulfo-hirudin was expressed.

Second, a control expression was run in which no sulfotyrosine wasadded. Subsequent MALDI-TOF analysis of the crude concentrated mediacontaining a mixture of all secreted proteins shows only a [M+H] peak of6578 Da corresponding to truncated protein resulting from TAG'salternative behavior as a stop codon (M_(theoretical)=6575 Da); no peakcorresponding to full-length protein was observed (see FIG. 6A). This isin contrast to expression in the presence of sulfotyrosine in which boththe truncated and full-length protein peaks are found in the massspectra at approximately equal intensities (see FIG. 6B), suggestingstrict dependence of amber suppression on the presence of sulfotyrosine.From these two experiments, it is concluded that the [M+H-80] signal inthe MALDI-TOF of sulfo-hirudin is solely attributable to SO₃ ⁻ cleavageduring the mass spectrometry, confirming that STyrRS charges its cognatetRNA exclusively with sulfotyrosine with no observable aminoacylation oftyrosine.

One should note that the similar intensities of the truncated andfull-length peaks in the mass spectra of crude sulfo-hirudin expressionmedia combined with the fact that expression of desulfo-hirudin yieldsapproximately twice as much protein as expression of sulfo-hirudin underthe same conditions suggest suppression for approximately half thetranslation events during the expression of sulfo-hirudin. One cantherefore infer that double suppression in our system will yieldapproximately 75% truncated and 25% full-length protein, assuming theabsence of amber suppression context effects. It is contemplated thatthe presence of truncated protein is due to low permeability of theanionic sulfotyrosine into E. coli cells, resulting in a decreasedpopulation of MjtRNA^(Tyr) _(CUA) charged with amino acid. In fact,expression of hirudin using the same system, but with the highlypermeable p-acetyl phenylalanine and its corresponding mutantsynthetase, yields incorporation of p-acetyl phenylalanine with nodetectable truncated protein (data not shown). A prodrug strategy todeliver sulfotyrosine may therefore eliminate the presence of truncatedprotein and increase yield.

Example 5 Characterization of Biological Activity of Genetically EncodedSulfo-Hirudin

To characterize the efficacy of the expressed sulfo-hirudin as ananticoagulant, the kinetics of thrombin inhibition were determined usinga fluorogenic enzyme assay based on the single progress curve methodpreviously reported in the literature (Cha, “Tight-bindinginhibitors—III. A new approach for the determination of competitionbetween tight-binding inhibitors and substrates—inhibition of adenosinedeaminase by coformycin,” Biochem Pharmacol 25:2695-2702 (1976); Komatsuet al., “CX-397, a novel recombinant hirudin analog having a hybridsequence of hirudin variants-1 and -3,” Biochem Biophys Res Commun196:773-779 (1993)). In this assay, 100 pM of either sulfo-hirudin ordesulfo-hirudin was mixed with 50 μM fluorogenic substrate to whichhuman α-thrombin was added to initiate the reaction. Cleavage of thefluorogenic substrate by thrombin, whose activity is inhibited todifferent degrees by sulfo-hirudin and desulfo-hirudin, resulted in aplot of fluorescence intensity over time (FIG. 3).

The exact concentrations of hirudin and sulfo-hirudin were determined bytitration against thrombin in a concentration range where 1:1 bindingcould be assumed. As follows from the tight-binding kinetics appropriateto hirudin (Stone and Hofsteenge, “Kinetics of the inhibition ofthrombin by hirudin,” Biochemistry 25:4622-4628 (1986)), theseexperimental data were fit to equation 1, yielding K_(i), k_(on), andk_(off) after manipulation of the extracted constants. This analysisafforded K_(i)'s for sulfo-hirudin and desulfo-hirudin of 26 fM and 307fM respectively, in agreement with literature reports (17). As expected,k_(on) for sulfo-hirudin (0.95×10⁸ M⁻¹s⁻¹) was greater than fordesulfo-hirudin (0.38×10⁸ M⁻¹s⁻¹), while k_(off) for sulfo-hirudin wassmaller (0.22×10⁻⁵ s⁻¹) than for desulfo-hirudin (1.18×10⁻⁵ s⁻¹). Thesethrombin inhibition kinetic constants derived from non-linear fitting ofprogress curves averaged over at least 3 readings with standarddeviations reported are shown in the table below.

K_(i) k_(on) × 10⁻⁸ (M⁻¹s⁻¹) k_(off) × 10⁵ (s⁻¹) Sulfo-hirudin  26 ± 9.80.95 ± 0.56 0.22 ± 0.06 Desulfo-hirudin 307 ± 72 0.38 ± 0.07 1.18 ± 0.45

The advantage of the higher affinity sulfo-hirudin over desulfo-hirudinshould be especially pronounced in the thrombin concentration rangeloosely bound by their respective K_(i)'s (Szedlacsek and Duggleby,“Kinetics of slow and tight-binding inhibitors,” Methods Enzymol249:144-180 (1995)). It is therefore interesting that the baselinephysiological steady-state concentration of active human thrombin fallswithin this range (Velan and Chandler, “Effects of surgical trauma andcardiopulmonary bypass on active thrombin concentrations and the rate ofthrombin inhibition in vivo,” Pathophysiol Haemost Thromb 33:144-156(2003)), suggesting a possible evolutionary impetus for sulfation innative leech hirudin. This observation should serve as a guide forpossible therapeutic applications for the genetically-encodedsulfo-hirudin (described herein) over the prevailing non-sulfatedrecombinant form.

The cotranslational incorporation of sulfotyrosine into proteins shouldmake possible the efficient expression of many more selectively sulfatedproteins in E. coli including antibodies, chemokine receptor motifs, andclotting factors, thereby facilitating structure-function studies aswell as the practical therapeutic application of sulfated proteins.Moreover, this in vivo strategy can be applied towards the constructionof sulfated antibody libraries and phage display of sulfated proteins,promising avenues inaccessible by the available methods of peptidesynthesis, native chemical ligation, and expressed protein ligation.Alternatively, it should be possible to extend this strategy to thedirect expression of tyrosine-sulfated proteins in eukaryotic organisms.

Detailed Methodology of Kinetic Characterization of Expressed HirudinSpecies

The release of 7-amino-4-methylcoumarin from 50 μMBoc-Asp(OBzl)-Pro-Arg-MCA as a result of thrombin activity was monitoredby measuring fluorescence intensity (excitation wavelength=365 nm;emission wavelength=450 nm) with a fluorescent plate reader (MolecularDevices SpectraMax Gemini). The enzyme reaction was done in triplicateand repeated thrice in 96-well plates at 37° C. in 50 mM Tris-HClbuffer, pH 7.8, containing 0.1% Polyethylene Glycol 6000 (Fluka), 100 mMNaCl, and 250 μg/mL HSA (Calbiochem). The Michaelis constant of thesubstrate under these conditions is 11.6 μM (Komatsu et al., “CX-397, anovel recombinant hirudin analog having a hybrid sequence of hirudinvariants-1 and -3,” Biochem Biophys Res Commun 196:773-779 (1993)).

The kinetic parameters of thrombin inhibition by the expressedsulfo-hirudin and desulfo-hirudin were extracted from non-linear fittingof progress curves obtained at 40 pM α-thrombin and 100 pM sulfo-hirudinor desulfo-hirudin using the single progress curve method, as previouslydescribed (Komatsu et al., “CX-397, a novel recombinant hirudin analoghaving a hybrid sequence of hirudin variants-1 and -3,” Biochem BiophysRes Commun 196:773-779 (1993)). According to the slow, tight-bindingcompetitive inhibition mechanism of hirudins, the product formation canbe described by equation 1 (Stone and Hofsteenge, “Kinetics of theinhibition of thrombin by hirudin,” Biochemistry 25:4622-4628 (1986);Cha, “Tight-binding inhibitors—III. A new approach for the determinationof competition between tight-binding inhibitors andsubstrates—inhibition of adenosine deaminase by coformycin,” BiochemPharmacol 25:2695-2702 (1976)):

$P = {{v_{s}t} + {\frac{\left( {1 - \gamma} \right)\left( {v_{0} - v_{s}} \right)}{\lambda \; \gamma}{\ln \left( \frac{1 - {\gamma \; ^{{- \lambda}\; t}}}{1 - \gamma} \right)}}}$

where P is the amount of product formed at time t, and v_(o) and v_(s)are the initial and steady-state velocities of the reaction. In equation1, v_(s), γ, and λ can be described by the following expressions:

$v_{s} = {v_{0}\left( \frac{E_{t} - I_{t} - K_{i}^{\prime} + Q}{2\; E_{t}} \right)}$$\gamma = \frac{K_{i}^{\prime} + E_{t} + I_{t} - Q}{K_{i}^{\prime} + E_{t} + I_{t} + Q}$λ = k_(on)Q, where$K_{i}^{\prime} = {K_{i}\left( {1 + \frac{S}{K_{m}}} \right)}$ and$Q = \sqrt{\left( {K_{i}^{\prime} + E_{t} + I_{t}} \right)^{2} - {4\; E_{t}I_{t}}}$

Using these equations, K_(i) and k_(on) were determined. The value ofk_(off) is the product of k_(on) and K_(i). Non-linear regressionfittings were calculated using the program GraphPad Prism.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. All publications, patents, patent applications, and/or otherdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

1. A translation system comprising: (a) a first unnatural amino acidthat is sulfotyrosine; (b) a first orthogonal aminoacyl-tRNA synthetase(O-RS); and (c) a first orthogonal tRNA (O-tRNA); wherein said firstO-RS preferentially aminoacylates said first O-tRNA with saidsulfotyrosine with an efficiency that is at least 50% of the efficiencyobserved for a translation system comprising said O-tRNA, saidsulfotyrosine, and an aminoacyl-tRNA synthetase comprising the aminoacid sequence of SEQ ID NO: 4, 6, 8 or
 10. 2. The translation system ofclaim 1, wherein said first O-RS is derived from a Methanococcusjannaschii aminoacyl-tRNA synthetase.
 3. The translation system of claim1, wherein said first O-RS is derived from a wild-type Methanococcusjannaschii tyrosyl-tRNA synthetase.
 4. The translation system of claim1, wherein said first O-RS comprises an amino acid sequence set forth inSEQ ID NO: 4, 6, 8 or 10, and conservative variants thereof.
 5. Thetranslation system of claim 1, wherein said first O-tRNA is an ambersuppressor tRNA.
 6. The translation system of claim 1, wherein saidfirst O-tRNA comprises or is encoded by a polynucleotide sequence setforth in SEQ ID NO:
 1. 7. The translation system of claim 1, furthercomprising a nucleic acid encoding a protein of interest, said nucleicacid comprising at least one selector codon, wherein said selector codonis recognized by said first O-tRNA.
 8. The translation system of claim7, further comprising a second O-RS and a second O-tRNA, wherein thesecond O-RS preferentially aminoacylates the second O-tRNA with a secondunnatural amino acid that is different from the first unnatural aminoacid, and wherein the second O-tRNA recognizes a selector codon that isdifferent from the selector codon recognized by the first O-tRNA.
 9. Thetranslation system of claim 1, wherein said system comprises a host cellcomprising said first unnatural amino acid, said first O-RS and saidfirst O-tRNA.
 10. The translation system of claim 9, wherein said hostcell is a eubacterial cell.
 11. The translation system of claim 10,wherein said eubacterial cell is an E. coli cell.
 12. The translationsystem of claim 9, wherein said host cell comprises a polynucleotideencoding said first O-RS.
 13. The translation system of claim 12,wherein said polynucleotide comprises a nucleotide sequence set forth inSEQ ID NO: 5, 7, 9 or
 11. 14. The translation system of claim 9, whereinsaid host cell comprises a polynucleotide encoding said first O-tRNA.15. A method for producing in a translation system a protein comprisingan unnatural amino acid at a selected position, the method comprising:(a) providing a translation system comprising: (i) a first unnaturalamino acid that is sulfotyrosine; (ii) a first orthogonal aminoacyl-tRNAsynthetase (O-RS); (iii) a first orthogonal tRNA (O-tRNA), wherein saidfirst O-RS preferentially aminoacylates said first O-tRNA with saidsulfotyrosine with an efficiency that is at least 50% of the efficiencyobserved for a translation system comprising said O-tRNA, saidsulfotyrosine, and an aminoacyl-tRNA synthetase comprising the aminoacid sequence of SEQ ID NO: 4, 6, 8 or 10; and, (iv) a nucleic acidencoding said protein, wherein said nucleic acid comprises at least oneselector codon that is recognized by said first O-tRNA; and, (b)incorporating said unnatural amino acid at said selected position insaid protein during translation of said protein in response to saidselector codon, thereby producing said protein comprising said unnaturalamino acid at the selected position.
 16. The method of claim 15, whereinsaid protein comprising an unnatural amino acid is sulfo-hirudin. 17.The method of claim 15, wherein said providing a translation systemcomprises providing a polynucleotide encoding said O-RS.
 18. The methodof claim 15, wherein said providing a translation system comprisesproviding an O-RS derived from a Methanococcus jannaschii aminoacyl-tRNAsynthetase.
 19. The method of claim 15, wherein said providing atranslation system comprises providing an O-RS derived from a wild-typeMethanococcus jannaschii tyrosyl-tRNA synthetase.
 20. The method ofclaim 15, wherein said providing a translation system comprisesproviding an O-RS comprising an amino acid sequence set forth in SEQ IDNO: 4, 6, 8 or 10, and conservative variants thereof.
 21. The method ofclaim 15, wherein said providing a translation system comprises mutatingan amino acid binding pocket of a wild-type aminoacyl-tRNA synthetase bysite-directed mutagenesis, and selecting a resulting O-RS thatpreferentially aminoacylates said O-tRNA with said unnatural amino acid.22. The method of claim 21, wherein said selecting step comprisespositively selecting and negatively selecting for said O-RS from a poolcomprising a plurality of resulting aminoacyl-tRNA synthetase moleculesfollowing site-directed mutagenesis.
 23. The method of claim 15, whereinsaid providing a translation system comprises providing a polynucleotideencoding said O-tRNA.
 24. The method of claim 15, wherein said providinga translation system comprises providing an O-tRNA that is an ambersuppressor tRNA.
 25. The method of claim 15, wherein said providing atranslation system comprises providing an O-tRNA that comprises or isencoded by a polynucleotide sequence set forth in SEQ ID NO:
 1. 26. Themethod of claim 15, wherein said providing a translation systemcomprises providing a nucleic acid comprising an amber selector codon.27. The method of claim 15, further wherein said protein comprises asecond unnatural amino acid that is different from said first unnaturalamino acid, and wherein said translation system further comprises asecond O-RS and a second O-tRNA, wherein the second O-RS preferentiallyaminoacylates the second O-tRNA with a second unnatural amino acid thatis different from the first unnatural amino acid, and wherein the second0-tRNA recognizes a selector codon in the nucleic acid that is differentfrom the selector codon recognized by the first O-tRNA.
 28. The methodof claim 15, wherein said providing a translation system comprisesproviding a host cell, wherein said host cell comprises said firstunnatural amino acid, said first O-RS, said first O-tRNA and saidnucleic acid, and wherein said incorporating step comprises culturingsaid host cell.
 29. The method of claim 28, wherein said providing ahost cell comprises providing a eubacterial host cell.
 30. The method ofclaim 29, wherein said providing a eubacterial host cell comprisesproviding an E. coli host cell.
 31. The method of claim 28, wherein saidproviding a host cell comprises providing a host cell comprising apolynucleotide encoding said O-RS.
 32. The method of claim 31, whereinsaid providing a host cell comprising a polynucleotide encoding saidO-RS step comprises providing a host cell comprising a polynucleotidecomprising a nucleotide sequence set forth in SEQ ID NO: 5, 7, 9 or 11.33. The method of claim 15, wherein said providing a translation systemcomprises providing a cell extract.
 34. A composition comprising apolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4,6, 8 or 10, or a conservative variant thereof.
 35. The composition ofclaim 34, wherein said conservative variant polypeptide aminoacylates acognate orthogonal tRNA (O-tRNA) with an unnatural amino acid with anefficiency that is at least 50% of the efficiency observed for atranslation system comprising said O-tRNA, said unnatural amino acid,and an aminoacyl-tRNA synthetase comprising the amino acid sequence ofSEQ ID NO: 4, 6, 8 or
 10. 36. A polynucleotide encoding the polypeptideof claim
 34. 37. The polynucleotide of claim 36, wherein saidpolynucleotide comprises the nucleotide sequence of SEQ ID NO: 5, 7, 9or
 11. 38. The composition of claim 34, where said composition comprisesa cell comprising the polypeptide.
 39. A vector comprising apolynucleotide of claim
 36. 40. An expression vector comprising apolynucleotide of claim
 36. 41. A cell comprising a vector, the vectorcomprising a polynucleotide of claim
 36. 42. A composition comprising apolynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:5, 7, 9 or 11.